Pentopyranosyl nucleic acid arrays, and uses thereof

ABSTRACT

The invention relates to a pentopyranosylnucleoside of the formula (I) or of the formula (II) 
                         
their preparation and use for the production of a therapeutic, diagnostic and/or electronic component.

This is a continuation of U.S. application Ser. No. 09/509,039 filedJul. 11, 2000, now U.S. Pat. No. 6,506,896 which in turn is a nationalstage application of international application PCT/EP98/05999, filedSep. 21, 1998, which in turn claims priority to German Application No.197 41 715.9, filed Sep. 22, 1997. All of the above applications areexpressly incorporated herein by reference.

The present invention relates to a pentopyranosylnucleoside of theformula (I) or of the formula (II)

its preparation and use for the production of an electronic component,in particular in the form of a diagnostic.

Pyranosylnucleic acids (p-NAs) are in general structural types which areisomeric to the natural RNA, in which the pentose units are present inthe pyranose form and are repetitively linked by phosphodiester groupsbetween the positions C-2′ and C-4′ (FIG. 1). “Nucleobase” is understoodhere as meaning the canonical nucleobases A, T, U, C, G, but also thepairs isoguanine/isocytosine and 2,6-diaminopurine/xanthine and, withinthe meaning of the present invention, also other purines andpyrimidines. p-NAs, namely the p-RNAs derived from ribose, weredescribed for the first time by Eschenmoser et al. (see Pitsch, S. etal. Helv. Chim. Acta 1993, 76, 2161; Pitsch, S. et al. Helv. Chim Acta1995, 78, 1621; Angew. Chem. 1996, 108, 1619–1623). They exclusivelyform so-called Watson-Crick-paired, i.e. purine-pyrimidine- andpurine-purine-paired, antiparallel, reversibly “melting”, quasilinearand stable duplexes. Homochiral p-RNA strands of the opposite sense ofchirality likewise pair controllably and are strictly non-helical in theduplex formed. This specificity, which is valuable for the constructionof supramolecular units, is associated with the relatively lowflexibility of the ribopyranose phosphate backbone and with the stronginclination of the base plane to the strand axis and the tendencyresulting from this for intercatenary base stacking in the resultingduplex and can finally be attributed to the participation of a2′,4′-cis-disubstituted ribopyranose ring in the construction of thebackbone. These significantly better pairing properties make p-NAspairing systems to be preferred compared with DNA and RNA for use in theconstruction of supramolecular units. They form a pairing system whichis orthogonal to natural nucleic acids, i.e. they do not pair with theDNAs and RNAs occurring in the natural form, which is of importance, inparticular, in the diagnostic field.

Eschenmoser et al. (1993, supra) has for the first time prepared ap-RNA, as shown in FIG. 2 and illustrated below.

In this context, a suitable protected nucleobase was reacted with theanomer mixture of the tetrabenzoylribopyranose by action ofbis(trimethylsilyl)acetamide and of a Lewis acid such as, for example,trimethylsilyl trifluoromethanesulphonate (analogously to H. Vorbrüggen,K. Krolikiewicz, B. Bennua, Chem. Ber. 1981, 114, 1234). Under theaction of base (NaOH in THF/methanol/water in the case of the purines;saturated ammonia in MeOH in the case of the pyrimidines), the acylprotected groups were removed from the sugar, and the product wasprotected in the 3′,4′-position under acidic catalysis withp-anisaldehyde dimethyl acetal. The diastereomer mixture was acylated inthe 2′-position, and the 3′,4′-methoxybenzylidene-protected 2′-benzoatewas deacetalized by acidic treatment, e.g. with trifluoroacetic acid inmethanol, and reacted with dimethoxytrityl chloride. The 2′→3′ migrationof the benzoate was initiated by treatment withp-nitrophenol/4-(dimethylamino)pyridine/triethylamine/pyridine/n-propanol.Almost all reactions were worked up by column chromatography. The keyunit synthesized in this way, the 4′-DMT-3′-benzoyl-1′-nucleobasederivative of the ribopyranose, was then partly phosphitylated andbonded to a solid phase via a linker.

In the following automated oligonucleotide synthesis, the carrier-bondedcomponent in the 4′-position was repeatedly acidically deprotected, aphosphoramidite was coupled on under the action of a coupling reagent,e.g. a tetrazole derivative, still free 4′-oxygen atoms were acetylatedand the phosphorus atom was oxidized in order thus to obtain theoligomeric product. The residual protective groups were then removed,and the product was purified and desalted by means of HPLC.

The described process of Eschenmoser et al. (1993, supra), however,shows the following disadvantages:

-   1. The use of non-anomerically pure tetrabenzoylpentopyranoses    (H. G. Fletcher, J. Am. Chem. Soc. 1955, 77, 5337) for the    nucleosidation reaction with nucleobases reduces the yields of the    final product owing to the necessity of rigorous chromatographic    cuts in the following working steps.-   2. With five reaction stages, starting from ribopyranoses which have    a nucleobase in the 1′-position, up to the protected 3′-benzoates,    the synthesis is very protracted and carrying-out on the industrial    scale is barely possible. In addition to the high time outlay, the    yields of monomer units obtained are low: 29% in the case of the    purine unit adenine, 24% in the case of the pyrimidine unit uracil.-   3. In the synthesis of the oligonucleotides,    5-(4-nitrophenyl)-1H-tetrazole is employed as a coupling reagent in    the automated p-RNA synthesis. The concentration of this reagent in    the solution of tetrazole in acetonitrile is in this case so high    that the 5-(4-nitrophenyl)-1H-tetrazole regularly crystallizes out    in the thin tubing of the synthesizer and the synthesis thus comes    to a premature end. Moreover, it was observed that the oligomers    were contaminated with 5-(4-nitrophenyl)-1-tetrazole.-   4. The described work-up of p-RNA oligonucleotides, especially the    removal of the base-labile protective groups with hydrazine    solution, is not always possible if there is a high thymidine    fraction in the oligomers.

A biomolecule, e.g. DNA or RNA, can be used for non-covalent linkingwith another biomolecule, e.g. DNA or RNA, if both biomolecules containsections which, as a result of complementary sequences of nucleobases,can bind to one another by formation of hydrogen bridges. Biomoleculesof this type are used, for example, in analytical systems for signalamplification, where a DNA molecule whose sequence is to be analysed ison the one hand to be immobilized by means of such a non-covalent DNAlinker on a solid support, and on the other hand is to be bonded to asignal-amplifying branched DNA molecule (bDNA)(see, for example, S.Urdea, Biol/Technol. 1994, 12, 926 or U.S. Pat. No. 5,624,802). Anessential disadvantage of the last-described systems is that to datethey are subject with respect to sensitivity to the processes fornucleic acid diagnosis by polymerase chain reaction (PCR) (K. Mullis,Methods Enzymol. 1987, 155, 335). This is to be attributed, inter alia,to the fact that the non-covalent bonding of the solid support to theDNA molecule to be analysed as well as the non-covalent bonding of theDNA molecule to be analysed does not always take place specifically; asa result of which a mixing of the functions “sequence recognition” and“non-covalent bonding” occurs.

The object of the present invention was therefore to provide novelbiomolecules and a process for their preparation in which theabove-described disadvantages can be avoided.

The use of p-NAs as an orthogonal pairing system which does notintervene in the DNA or RNA pairing process solves this problemadvantageously, as a result of which the sensitivity of the analyticalprocesses described can be markedly increased.

One subject of the present invention is therefore the use ofpentopyranosylnucleotides or pentopyranosylnucleic acids preferably inthe form of a conjugate comprising a pentopyranosylnucleotide or apentopyranosylnucleic acid and a biomolecule for the production of anelectronic component, in particular in the form of a diagnostic.

Conjugates within the meaning of the present invention are covalentlybonded hybrids of p-NAs and other biomolecules, preferably a peptide,protein or a nucleic acid, for example an antibody or a functionalmoiety thereof or a DNA and/or RNA occurring in its natural form.Functional moieties of antibodies are, for example, Fv fragments (Skerra& Plückthun (1988) Science 240, 1038), single-chain Fv fragments (scFv;Bird et al. (1988), Science 242, 423; Huston et al. (1988) Proc. Natl.Acad. Sci. USA, 85, 5879) or Fab fragments (Better et al. (1988) Science240, 1041).

Biomolecule within the meaning of the present invention is understood asmeaning a naturally occurring substance or a substance derived from anaturally occurring substance.

In a preferred embodiment, they are in this case p-RNA/DNA or p-RNA/RNAconjugates.

Conjugates are preferably used when the functions “sequence recognition”and “non-covalent bonding” must be realized in a molecule, since theconjugates according to the invention contain two pairing systems whichare orthogonal to one another.

p-NAs and in particular the p-RNAs form stable duplexes with one anotherand in general do not pair with the DNAs and RNAs occurring in theirnatural form. This property makes p-NAs preferred pairing systems.

Such pairing systems are supramolecular systems of non-covalentinteraction, which are distinguished by selectivity, stability andreversibility, and their properties are preferably influencedthermodynamically, i.e. by temperature, pH and concentration. On.account of their selective properties, such pairing systems can also beused, for example, as “molecular adhesive” for the bringing together ofdifferent metal clusters to give cluster associations having potentiallynovel properties [see, for example, R. L. Letsinger, et al., Nature1996, 382, 607–9; P. G. Schultz et al., Nature 1996, 382, 609–11].Consequently, the p-NAs are also suitable for use in the field ofnanotechnology, for example for the production of novel materials,diagnostics and therapeutics and also microelectronic, photonic oroptoelectronic components and for the controlled bringing together ofmolecular species to give supramolecular units, such as, for example,for the (combinatorial) synthesis of protein assemblies [see, forexample, A. Lombardi, J. W. Bryson, W. F. DeGrado, Biomoleküls (Pept.Sci.) 1997, 40, 495–504], as p-NAs form pairing systems which arestrongly and thermodynamically controllable. A further applicationtherefore especially arises in the diagnostic and drug discovery fielddue to the possibility of providing functional, preferably biologicalunits, such as proteins or DNA/RNA sections, with a p-NA code which doesnot interfere with the natural nucleic acids (see, for example, WO93/20242).

Both sequential and convergent processes are suitable for thepreparation of conjugates.

In a sequential process, for example after automated synthesis of ap-RNA oligomer has taken place directly on the same synthesizer—afterreadjustment of the reagents and of the coupling protocol—a DNAoligonucleotide, for example, is additionally synthesized. This processcan also be carried out in the reverse sequence.

In a convergent process, for example, p-RNA oligomers havingamino-terminal linkers and, for example, DNA oligomers having, forexample, thiol linkers are synthesized in separate operations. Aniodoacetylation of the p-RNA oligomer and the coupling of the two unitsaccording to protocols known from the literature (T. Zhu et al.,Bioconjug. Chem. 1994, 5, 312) is then preferably carried. out.Convergent processes prove to be particularly preferred on account oftheir flexibility.

The term conjugate within the meaning of the present invention is alsounderstood as meaning so-called arrays. Arrays are arrangements ofimmobilized recognition species which, especially in analysis anddiagnosis, play an important role in the simultaneous determination ofanalytes. Examples are peptide arrays (Fodor et al., Nature 1993, 364,555) and nucleic acid arrays (Southern et al. Genomics 1992, 13, 1008;Heller, U.S. Pat. No. 5,632,957). A higher flexibility of these arrayscan be achieved by binding the recognition species to codingoligonucleotides and the associated, complementary strands to certainpositions on a solid carrier. By applying the coded recognition speciesto the “anti-coded” solid carrier and adjustment of hybridizationconditions, the recognition species are non-covalently bonded to thedesired positions. As a result, various types of recognition species,such as, for example, DNA sections, antibodies, can only be arrangedsimultaneously on a solid carrier by use of hybridization conditions(see FIG. 3). The prerequisite for this, however, are codons andanticodons which are extremely strong and selective—in order to keep thecoding sections as short as possible—and do not interfere with naturalnucleic acid necessary. p-NAs, preferably p-RNAs, are particularlyadvantageously suitable for this.

The term “carrier” within the meaning of the present invention isunderstood as meaning material, in particular chip material, which ispresent in solid or alternatively gelatinous form. Suitable carriermaterials are, for example, ceramic, metal, in particular noble metal,glasses, plastics, crystalline materials or thin layers of the carrier,in particular of the materials mentioned, or (bio)molecular filamentssuch as cellulose, structural proteins.

The present invention therefore also relates to the use ofpentopyranosylnucleic acids, preferably ribopyranosylnucleic acids forencoding recognition species, preferably natural DNA or RNA strands orproteins, in particular antibodies or functional moieties of antibodies.These can then be hybridized with the appropriate codons on a solidcarrier according to FIG. 3. Thus arrays which are novel anddiagnostically useful can always be built up in the desired positions ona solid carrier which is equipped with codons in the form of an arrayonly by adjustment of hybridization conditions using combinations ofrecognition species which are always novel. If the analyte, for examplea biological sample such as serum or the like, is then applied, thespecies to be detected are bonded to the array in a certain patternwhich is then recorded indirectly (e.g. by fluorescence labelling of therecognition species) or directly (e.g. by impedance measurement at thelinkage point of the codon). The hybridization is then eliminated bysuitable conditions (temperature, salts, solvents, electrophoreticprocesses) so that again only the carrier having the codons remains.This is then again loaded with other recognition species and is used,for example, for the same analyte for the determination of anothersample. The always new arrangement of recognition species in the arrayformat and the use of p-NAs as pairing systems is particularlyadvantageous compared with other systems, see, for example, WO 96/13522(see 16, below).

In a preferred embodiment, the pentopyranosylnucleoside is a compound ofthe formula (I)

in which

-   R¹ is equal to H, OH, Hal where Hal is equal to Br or-   Cl or a radical selected from-   or—O—P[N(i-Pr)₂]—(OCH₂CH₂CN)    where i-Pr is equal to isopropyl, R², R³ and R⁴ independently of one    another, identically or differently, are in each case H, Hal where    Hal is equal to Br or Cl, NR⁵R⁶, OR⁷, SR⁸, ═O, C_(n)H_(2n+1) where n    is an integer from 1–12, preferably 1–8, in particular 1–4, a    β-eliminable group, preferably a group of the formula —OCH₂CH₂R¹⁸    where R¹⁸ is equal to a cyano or p-nitrophenyl radical or a    fluorenylmethyloxycarbonyl (Fmoc) radical, or (C_(n)H_(2n))NR¹⁰R¹¹    where R¹⁰R¹¹ is equal to H, C_(n)H_(2n+1) or R¹⁰R¹¹ linked via a    radical of the formula

in which R¹², R¹³, R¹⁴ and R¹⁵ independently of one another, identicallyor differently, are in each case H, OR⁷, where R⁷ has the abovementionedmeaning, or C_(n)H_(2n+1), or C_(n)H_(2n−1), where n has theabovementioned meaning, and

-   R⁵, R⁶, R⁷ and R⁸ independently of one another, identically or    differently, is in each case H, C_(n)H_(2n+1), or C_(n)H_(2n−1),    where n has the abovementioned meaning, —C(O)R⁹ where R⁹ is equal to    a linear or branched, optionally substituted alkyl or aryl radical,    preferably a phenyl radical,-   X, Y and Z independently of one another, identically or differently,    is in each case ═N—, ═C(R¹⁶)— or —N(R¹⁷)— where R¹⁶ and R¹⁷    independently of one another, identically or differently, is in each    case H or C_(n)H_(2n+1) or (C_(n)H_(2n))NR¹⁰R¹¹ having the    abovementioned meanings, and S_(c1) and S_(c2) independently of one    another, identically or differently, is in each case H or a    protective group selected from an acyl, trityl or allyloxycarbonyl    group, preferably a benzoyl or 4,4′-dimethoxytrityl (DMT) group,    or of the formula (II)

in which R^(1′) is equal to H, OH, Hal where Hal is equal to Br or Cl,or a radical selected from

-   or —O—P[N(i-Pr)₂]—(OCH₂CH₂CN)    where i-Pr is equal to isopropyl,-   R^(2′), R^(3′) and R^(4′) independently of one another, identically    or differently, is in each case H, Hal where Hal is equal to Br or    Cl ═O, C_(n)H_(2n+1) or C_(n)H_(2n−1), a β-eliminable group,    preferably a group of the formula —OCH₂CH₂R¹⁸ where R¹⁸ is equal to    a cyano or p-nitrophenyl radical or a fluorenylmethyloxycarbonyl    (Fmoc) radical or (C_(n)H_(2n))NR^(10′)R^(11′), where R^(10′),    R^(11′), independently of one another has the abovementioned meaning    of R¹⁰ or R¹¹, and

X′, is in each case ═N—, ═C(R^(16′))— or —N(R¹⁷)—, where R^(16′) andR^(17′) independently of one another have the abovementioned meaning ofR¹⁶ or R¹⁷, and S_(c1′) and S_(c2′) have the abovementioned meaning ofS_(a1) and S_(c2).

The pentopyranosylnucleoside is in general a ribo-, arabino-, lyxo-and/or xylopyranosylnucleoside, preferably a ribopyranosylnucleoside,where the pentopyranosyl moiety can be in the D configuration, but alsoin the L configuration.

Customarily, the pentopyranosylnucleoside according to the invention isa pentopyranosylpurine,-2,6-diaminopurine, -6-purinethiol, -pyridine,-pyrimidine, -adenosine, -guanosine, -isoguanosine, -6-thioguanosine,-xanthine, -hypoxanthine, -thymidine, -cytosine, -isocytosine, -indole,-tryptamine, -N-phthaloyltryptamine, -uracil, -caffeine, -theobromine,-theophylline, -benzotriazole or -acridine, in particular apentopyranosylpurine, -pyrimidine, -adenosine, -guanosine, -thymidine,-cytosine, -tryptamine, -N-phthalotryptamine or -uracil.

The compounds also include pentopyranosylnucleosides which can be usedas linkers, i.e. as compounds having functional groups which can bondcovalently to biomolecules, such as, for example, nucleic acidsoccurring in their natural form or modified nucleic acids, such as DNA,RNA but also p-NAs, preferably pRNAs. This is surprising, as no linkersare yet known for p-NAs.

For example, these include pentopyranosylnucleosides in which R², R³,R⁴, R^(2′), R^(3′) and/or R^(4′) is a 2-phthalimidoethyl or allyloxyradical. Preferred linkers according to the present invention are, forexample, uracil-based linkers in which the 5-position of the uracil haspreferably been modified, e.g. N-phthaloylaminoethyluracil, but alsoindole-based linkers, preferably tryptamine derivatives, such as, forexample, N-phthaloyltryptamine.

Surprisingly, by means of the present invention more easily handleablepentopyranosyl-N,N-diacylnucleosides, preferably purines, in particularadenosine, guanosine or 6-thioguanosine, are also made available, whosenucleobase can be completely deprotected in a simple manner. Theinvention therefore also includes pentopyranosylnucleosides in which R²,R³, R⁴, R^(2′), R^(3′) and/or R^(4′) is a radical of the formula—N[C(O)R⁹]₂, in particular N⁶,N⁶-dibenzoyl-9-(β-D-ribopyranosyl)-adenosine.

It is furthermore surprising that the present invention makes availablepentopyranosylnucleosides which carry a protective group, preferably aprotective group which can be removed by base or metal catalysis, inparticular an acyl group, particularly preferably a benzoyl group,exclusively on the 3′-oxygen atom of the pentopyranoside moiety. Thesecompounds serve, for example, as starting substances for the directintroduction of a further protective group, preferably of an acid- orbase-labile protective group, in particular of a trityl group,particularly preferably a dimethoxytrityl group, onto the 4′-oxygen atomof the pentopyranoside moiety without additional steps which reduce theyield, such as, for example, additional purification steps.

Moreover, the present invention makes availablepentopyranosylnucleosides which carry a protective group, preferably anacid- or base-labile protective group, in particular a trityl group,particularly preferably a dimethoxytrityl group, exclusively on the4′-oxygen atom of the pentopyranoside moiety. These compounds too serve,for example, as starting substances for the direct introduction of afurther protective group, preferably of a protective group which can beremoved by base or metal catalysis, in particular of an acyl group,particularly preferably of a benzoyl group, e.g. on the 2′-oxygen atomof the pentopyranoside moiety, without additional steps which reduce theyield, such as, for example, additional purification steps.

In general, the pentopyranosidenucleosides according to the inventioncan be reacted in a so-called one-pot reaction, which increases theyields and is therefore particularly advantageous.

The following compounds are preferred examples of thepentopyranosylnucleosides according to the invention:

-   A) [2′,4′-Di-O-Benzoyl)-β-ribopyranosyl]nucleosides, in particular a    [2′,4′-di-O-benzoyl)-β-ribopyranosyl]-adenine, -guanine, -cytosine,    -thymidine, -uracil, -xanthine or -hypoxanthine, and an    N-benzoyl-2′,4′-di-O-benzoylribopyranosylnucleoside, in particular    an -adenine, -guanine or -cytosine, and an    N-isobutyroyl-2′,4′-di-O-benzoylribopyranosylnucleoside, in    particular an -adenine, -guanine or -cytosine, and an    O⁶-(2-cyanoethyl)-N²-isobutyroyl-2′,4′-di-O-benzoylribopyranosylnucleoside,    in particular a -guanine, and an O⁶    (2-(4-nitrophenyl)ethyl)-N²-isobutyroyl-2,4′-di-O-benzoylribopyranosylnucleoside,    in particular a -guanine.-   B) β-Ribopyranosylnucleosides, in particular a    β-ribopyranosyladenine, -guanine, -cytosine, -thymidine or -uracil,    -xanthine or hypoxanthine, and an N-benzoyl-, N-isobutyroyl-,    O⁶-(2-cyanoethyl)- or    O⁶-(2-(4-nitrophenyl)ethyl)-N²-isobutylroyl-β-ribopyranosylnucleoside.-   C) 4′-DMT-pentopyranosylnucleosides, preferably a    4′-DMT-ribopyranosylnucleoside, in particular a    4′-DMT-ribopyranosyladenine, -guanine, -cytosine, -thymidine,    -uracil, -xanthine or -hypoxanthine, and an    N-benzoyl-4′-DMT-ribopyranosylnucleoside, in particular an    N-benzoyl-4′-DMT-ribopyranosyladenine, -guanine or -cytosine, and an    N-isobutyroyl-4′-DMT-ribopyranosylnucleoside, in particular an    N-isobutyroyl-4′-DMT-ribopyranosyladenine, -guanine or -cytosine and    an O⁶-(2-cyanoethyl)-N²-isobutyroyl-4′-DMT-ribopyranosylnucleoside,    in particular an    O⁶-(2-cyanoethyl)-N²-isobutyroyl-4′-DMT-ribopyranosylguanine, and an    O⁶-2-(-4-nitrophenyl)ethyl)-N²-isobutyroyl-4′-DMT-ribopyranosylnucleoside,    in particular an    O⁶-(2-(-4-nitrophenyl)ethyl)-N²-isobutyroyl-4′-DMT-ribopyranosylguanine.-   D) β-Ribopyranosyl-N,N′-dibenzoyladenosine or    β-ribopyranosyl-N,N′-dibenzoylguanosine.

Suitable precursors for the oligonucleotide synthesis are, for example,4′-DMT-pentopyranosylnucleoside-2′-phosphitamide/-H-phosphonate,preferably a4′DMT-ribopyranosylnucleoside-2′-phosphitamide/-H-phosphonate, inparticular a 4′-DMT-ribopyranosyladenine-, -guanine-, -cytosine-,-thymidine-, -xanthine-, hypoxanthine-, or-uracil-2′-phosphitamide/-H-phosphonate and anN-benzoyl-4′-DMT-ribopyranosyladenine-, -guanine- or-cytosine-2′-phosphitamide/-H-phosphonate and anN-isobutylroyl-4′-DMT-ribopyranosyladenine-, -guanine- or-cytosine-2′-phosphitamide/-H-phosphonate,O⁶-(2-cyanoethyl)-4′-DMT-ribopyranosylguanine-, -xanthine-,-hypoxanthine-2′-phosphitamide/-H-phosphonate orO⁶-(2-(4-nitrophenyl)ethyl)-N²-isobutyroyl-4′-DMT-ribopyranosylguanine,and for the coupling to the solid carrier, for example,4′-DMT-pentopyranosylnucleoside-2′-succinate, preferably a4′-DMT-ribopyranosylnucleoside-2′-succinate, in particular a4′DMT-ribopyranosyladenine-, -gaunine-, -cytosine-, thymidine-,-xanthine-, -hypoxanthine- or -uracil-2′-succinate and anN-benzoyl-4′-DMT-ribopyranosyladenine-, -guanine- or-cytonsine-2′-succinate and anN-isobutyroyl-4′-DMT-ribopyranosyladenine-, -guanine- or-cytosine-2′-succinate, O-(2-cyanoethyl)-4′-DMT-ribopyranosylguaninesuccinate and anO⁶-(2-(4-nitrophenyl)ethyl)-N²-isobutyroyl-4′-DMT-ribopyranosylguanine-2′-succinate.

The pentapyranosylnucleosides can be particularly advantageouslyprepared according to the present invention in that, starting from theunprotected pentopyranoside,

-   (a) in a first step the 2′-, 3′- or 4′-position of the    pentopyranoside is first protected, and preferably-   (b) in a second step the other position is protected on the 2′-, 3′-    or 4′-position.

This process is not restricted to the nucleobases described in the citedliterature, but can surprisingly be carried out successfully using alarge number of natural and synthetic nucleobases. Moreover, it isparticularly surprising that the process according to the invention canbe carried out in high yields and with a time saving of on average 60%in comparison with the process known from the literature, which isparticularly advantageous for industrial application. In addition, usingthe process according to the invention the purification steps necessaryin the process described in the literature, e.g. chromatographicintermediate purifications, are not necessary and the reactions can insome cases be carried out as a so-called one-pot reaction, whichmarkedly increases the space/time yields.

In a particular embodiment, in the case of a 2′-protected position arearrangement of the protective group from the 2′-position to the3′-position takes place, which in general is carried out in the presenceof a base, in particular in the presence of N-ethyldiisopropylamineand/or triethylamine. According to the present invention, this reactioncan be carried out particularly advantageously in the same reactioncontainer as the one-pot reaction.

In a further preferred embodiment, the pyranosylnucleoside is protectedby a protective group S_(c1), S_(c2), S_(c1′) or S_(c2′) which isacid-labile, base-labile or can be removed with metal catalysis, theprotective groups S_(c1) and S_(c1′) preferably being different from theprotective groups S_(c2) and S_(c2′).

In general, the protective groups mentioned are an acyl group,preferably an acetyl, benzoyl, nitrobenzoyl and/or methoxybenzoyl group,trityl groups, preferably a 4,4′-dimethoxytrityl (DMT) group or aβ-eliminable group, preferably a group of the formula —OCH₂CH₂R¹⁸ whereR¹⁸ is equal to a cyano or p-nitrophenyl radical or afluorenylmethyloxycarbonyl (Fmoc) group.

It is particularly preferred if the 2′- or 3′-position is protected by aprotective group which is base-labile or can be removed with metalcatalysis, preferably by an acyl group, in particular by an acetyl,benzoyl, nitrobenzoyl and/or methoxybenzoyl group, and/or the4′-position is protected by an acid- or base-labile protective group,preferably by a trityl and/or Fmoc group, in particular by a DMT group.

Unlike the process known from the literature, this process consequentlymanages without acetal protective groups, such as acetals or ketals,which avoids additional chromatographic intermediate purifications andconsequently allows the reactions to be carried out as one-pot reactionswith surprisingly high space/time yields.

The protective groups mentioned are preferably introduced at lowtemperatures, as by this means they can be introduced surprisinglyselectively.

Thus, for example, the introduction of a benzoyl group takes place byreaction with benzoyl chloride in pyridine or in a pyridine/methylenechloride mixture at low temperatures. A DMT group can be introduced, forexample, by reaction with DMTCl in the presence of a base, e.g. ofN-ethyldiisopropylamine (Hünig's base), and, for example, of pyridine,methylene chloride or a pyridine/methylene chloride mixture at roomtemperature.

It is also advantageous if after the acylation and/or after therearrangement of the 2′- to the 3′-position which is optionally carriedout, the reaction products are purified by chromatography. Purificationafter the tritylation is not necessary according to the processaccording to the invention, which is particularly advantageous.

The final product, if necessary, can additionally be further purified bycrystallization.

For the preparation of a ribopyranosylnucleoside preferably first

-   (a) a protected nucleobase is reacted with a protected ribopyranose,    then-   (b) the protective groups are removed from the ribopyranosyl moiety    of the product from step (a), and then-   (c) the product from step (b) is reacted according to the process    described above in greater detail.

In this connection, in order to avoid further time- andmaterial-consuming chromatography steps, it is advantageous only toemploy anomerically pure protected pentopyranoses, such as, for example,tetrabenzoylpentopyranoses, preferably β-tetrabenzoylribopyranoses (R.Jeanloz, J. Am. Chem. Soc. 1948, 70, 4052).

In a further embodiment, a linker according to formula (II), in whichR^(4′) is (C_(n)H_(2n))NR^(10′)R^(11′) and R^(10′)R^(11′) is linked bymeans of a radical of the formula (III) having the meaning alreadydesignated, is advantageously prepared by the following process:

-   (a) a compound of the formula (II) where R^(4′) is equal to    (C_(n)H_(2n))OS_(c3) or (C_(n)H_(2n))Hal, in which n has the    abovementioned meaning, S_(c3) is a protective group, preferably a    mesylate group, and Hal is chlorine or bromine, is reacted with an    azide, preferably in DMF, then-   (b) the reaction product from (a), is preferably reduced with    triphenylphosphine, e.g. in pyridine, then-   (c) the reaction product from (b) is reacted with an appropriate    phthalimide, e.g. N-ethoxycarbonylphthalimide, and-   (d) the reaction product from (c) is reacted with an appropriate    protected pyranose, e.g. ribose tetrabenzoate, and finally-   (e) the protected groups are removed, for example with methylate,    and-   (f) the further steps are carried out as already described above.

In addition, indole derivatives as linkers have the advantage of theability to fluoresce and are therefore particularly preferred fornanotechnology applications in which it may be a matter of detectingvery small amounts of substance. Thus indole-1-ribosides have alreadybeen described in N. N. Suvorov et al., Biol. Aktivn. Soedin., Akad.Nauk SSSR 1965, 60 and Tetrahedron 1967, 23, 4653. However, there is noanalogous process for preparing 3-substituted derivatives. In general,their preparation takes place via the formation of an aminal of theunprotected sugar component and an indoline, which is then convertedinto the indole-1-riboside by oxidation. For example,indole-1-glucosides and -1-arabinosides have been described (Y. V.Dobriynin et al. Khim.-Farm Zh. 1978, 12, 33), whose 3-substitutedderivatives were usually prepared by means of Vielsmeier's reaction.This route for the introduction of aminoethyl units into the 3-positionof the indole is too complicated, however, for industrial application.

In a further preferred embodiment, a linker according to formula (I), inwhich X and Y independently of one another, identically or differently,are in each case ═C(R¹⁶) where R¹⁶ is equal to H or C_(n)H_(2n) and Z═C(R¹⁶)— where R¹⁶ is equal to (C_(n)H_(2n))NR¹⁰R¹¹ is thereforeadvantageously prepared by the following process:

-   (a) the appropriate indoline, e.g. N-phthaloyltryptamine, is reacted    with a pyranose, e.g. D-ribose, to give the nucleoside triol, then-   (b) the hydroxyl groups of the pyranosyl moiety of the product    from (a) are preferably protected with acyl groups, e.g. by means of    acetic anhydride, then-   (c) the product from (b) is oxidized, e.g. by    2,3-dichloro-5,6-dicyanoparaquinone, and-   (d) the hydroxyl protective groups of the pyranosyl moiety of the    product from (c) are removed, for example, by means of methylate and    finally-   (e) the further steps as already described above are carried out.

This process, however, cannot only be used in the case of ribopyranoses,but also in the case of ribofuranoses and 2′-deoxyribofuranoses or2′-deoxyribopyranoses, which is particularly advantageous. Thenucleosidation partner of the sugar used is preferably tryptamine, inparticular N-acyl derivatives of tryptamine, especiallyN-phthaloyltryptamine.

In a further embodiment, the 4′-protected, preferably, the3′,4′-protected pentopyranosylnucleosides are phosphitylated in afurther step or bonded to a solid phase.

Phosphitylation is carried out, for example, by means of monoallylN-diisopropylchlorophosphoramidite in the presence of a base, e.g.N-ethyldiisopropylamine or by means of phosphorus trichloride andimidazole or tetrazole and subsequent hydrolysis with addition of base.In the first case, the product is a phosphoramidite and in the secondcase an H-phosphonate. The bonding of a protectedpentopyranosylnucleoside according to the invention to a solid phase,e.g. “long-chain alkylamino-controlled pore glass” (CPG, Sigma Chemie,Munich) can be carried out, for example, as described in Eschenmoser etal. (1993).

The compounds obtained serve, for example, for the preparation ofpentopyranosylnucleic acids, in which, preferably

-   (a) in a first step a protected pentopyranosylnucleoside is bonded    to a solid phase as already described above and-   (b) in a second step the 3′-,4′-protected pentopyranosylnucleoside    bonded to a solid phase according to step (a) is lengthened by a    phosphitylated 3′-,4′-protected pentopyranosylnucleoside and then    oxidized, for example, by an aqueous iodine solution, and-   (c) step (b) is repeated with identical or different phosphitylated    3′-,4′-protected pentopyranosylnucleosides until the desired    pentopyranosylnucleic acid is present.

Acidic activators such as pyridinium hydrochloride, preferablybenzimidazolium triflate, are suitable as a coupling reagent whenphosphoramidites are employed, preferably after recrystallizing inacetonitrile and after dissolving in acetonitrile, as in contrast to5-(4-nitrophenyl)-1H-tetrazole as a coupling reagent no blockage of thecoupling reagent lines and contamination of the product takes place.

Arylsulphonyl chlorides, diphenyl chlorophosphate, pivaloyl chloride oradamantoyl chloride are particularly suitable as a coupling reagent whenH-phosphonates are employed.

Furthermore, it is advantageous by means of addition of a salt, such assodium chloride, to the protective-group-removing hydrazinolysis ofoligonucleotides, in particular of p-NAs, preferably of p-RNAs, toprotect pyrimidine bases, especially uracil and thymine, fromring-opening, which would destroy the oligonucleotide. Allyloxy groupscan preferably be removed by palladium [Pd(0)] complexes, e.g. beforehyrazinolysis.

In a further particular embodiment, pentofuranosylnucleosides, e.g.adenosine, guanosine, cytidine, thymidine and/or uracil occurring intheir natural form, can also be incorporated in step (a) and/or step(b), which leads, for example, to a mixed p-NA-DNA or p-NA-RNA.

In another particular embodiment, in a further step an allyloxy linkerof the formulaS_(c4)NH(C_(n)H_(2n))CH(OPS_(c5)S_(c6))C_(n)H_(2n)S_(c7)  (IV)in which S_(c4) and S_(c7) independently of one another, identically ordifferently, are in each case a protective group in particular selectedfrom Fmoc and/or DMT,

-   S_(c5) and S_(c6) independently of one another, identically or    differently, are in each case an allyloxy and/or diisopropylamino    group, can be incorporated. n has the meaning already mentioned    above.    A particularly preferred allyloxy linker is    (2-(S)-N-Fmoc-O¹-DMT-O²-allyloxydiisopropylaminophosphinyl-6-amino-1,2-hexanediol).

Starting from, for example, lysine, in a few reaction stepsamino-terminal linkers can thus be synthesized which carry both anactivatable phosphorus compound and an acid-labile protective group,such as DMT, and can therefore easily be used in automatableoligonucleotide synthesis (see, for example, P. S. Nelson et al.,Nucleic Acid Res. 1989, 17, 7179; L. J. Arnold et al., WO 8902439). Therepertoire was extended in the present invention by means of alysine-based linker, in which instead of the otherwise customarycyanoethyl group on the phosphorus atom an allyloxy group wasintroduced, and which can therefore be advantageously employed in theNoyori oligonucleotide method (R. Noyori, J. Am. Chem. Soc. 1990, 112,1691–6).

A further subject of the present invention also relates to an electroniccomponent in particular in the form of a diagnostic, comprising anabove-described pentopyranosylnucleoside or a pentopyranosylnucleosidein the form of a conjugate, and a process for the preparation of aconjugate, in which a pentopyranosylnucleoside or apentopyranosylnucleic acid is combined with a biomolecule, as alreadydescribed in detail above.

The following figures and examples are intended to describe theinvention in greater detail, without restricting it.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a section of the structure of RNA in its naturallyoccurring form (left) and in the form of a p-NA (right).

FIG. 2 schematically shows the synthesis of ap-ribo(A,U)-oligonucleotide according to Eschenmoser et al (1993).

FIG. 3 schematically shows an arrangement of immobilized recognitionstructures (arrays) on a solid carrier.

EXAMPLES Example 1 Synthesis of1-{3′-O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}thymine

First 4′-substitution, then 2′-substitution, then migration reaction:

51.6 g (200 mmol) of 1-(β-D-ribopyranosyl)thymine A were dissolved in620 ml of anhydrous pyridine under an argon atmosphere, 71.4 ml (2.1eq.) of N-ethyldiiosopropylamine and 100 g of molecular sieve (4 Å) wereadded and the mixture was stirred for 15 min using a KPG stirrer. 92 g(272 mmol; 1.36 eq.) of dimethoxytrityl chloride (DMTCl) were dissolvedin 280 ml (freshly distilled from solid NaHCO₃) of chloroform and thissolution was added dropwise to the triol solution at −6 to −50° C. inthe course of 30 min. It was stirred at this temp. for 1 h, then stirredovernight at room temperature (RT.), cooled again, and a further 25 g(74 mmol; 0.37 eq.) of DMTCl in 70 ml of chloroform were added. Themixture was allowed to come to RT. and was stirred for 4 h.

A small sample was taken, subjected to aqueous work-up andchromatographed in order to obtain the analytical data of the1-{4′-O-(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}thymine:

¹H-NMR (300 MHz, CDCl3): 1.70 (bs, 2H, OH) ; 1.84 (d, 3H, Me); 2.90 (bs,1H, OH); 3.18, 3.30 (2m, 2H, H(5′)), 3.62 (bs, 1H, H(3′)); 3.70–3.82 (m,8H, 2 OMe, H(4′), H(2′)); 5.75 (d, J=9.5 Hz, 1H, H(1′)), 6.85 (m, 4H,Harom); 6.96 (m, 1H, Harom), 7.20 (m, 9H, Harom, H(6)), 8.70 (bs, 1H,H(3).)

The reaction mixture was treated with 2.46 g (20.5 mmol; 0.1 eq.) of4-dimethylaminopyridine (DMAP), cooled to −6° C. and 27.9 ml (0.24 mol;1.2 eq.) of benzoyl chloride (BzCl) in 30 ml of pyridine were addeddropwise between −6 and −1° C. in the course of 15 min and the mixturewas stirred for 10 min. To complete the reaction, a further 2.8 ml (24mmol; 0.12 eq.) of BzCl were in each case added with cooling at aninterval of 25 min and the mixture was finally stirred for 20 min.

460 ml of anhydrous pyridine, 841 ml (11.2 mol; 56 eq.) of n-propanol,44 g (0.316 mol; 1.58 eq.) of p-nitrophenol, 21.7 g (0.18 mol; 0.9 eq.)of DMAP and 136 ml (0.8 mol; 4 eq.) of N-ethyldiisopropylamine were thenadded at RT. and the mixture was stirred at 61–63° C. for 48 h. Themixture was then allowed to stand at RT. for 60 h. The reaction mixturewas again heated to 61–63° C. for 24 h, cooled to RT. and concentratedon a Rotavapor. The residue was taken up in 2 l of ethyl acetate, themolecular sieve was filtered off, the org. phase was extracted threetimes with 1 l of water each time and extracted once by stirring with1.2 l of 10% strength citric acid and the org. phase was again separatedoff, extracted once with 1 l of water and finally with 1 l of saturatedNaHCO₃ solution. The org. phase was dried using sodium sulphate,filtered and concentrated (220 g of residue).

The residue was first filtered through silica gel 60 (20×10 cm) using astep gradient of heptane/ethyl acetate, 1:1 to 0.1) for prepurification,then chromatographed on silica gel 60 (30×10 cm; step gradient ofdichloromethane/ethyl acetate, 1:0 to 1:1).

The following were obtained:

-   40 g of non-polar fractions-   52.9 g of    1-{3′-O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl)-thymine    B-   34.5 g of impure B-   3.4 g of polar fractions

The impure fraction was chromatographed again (SG 60, 45×10 cm;dichloromethane/ethyl acetate, 3:1) and yielded a further 11.3 g of B.

Total yield: 64.2 g (97 mmol) of B, i.e. 48% yield. ¹H-NMR corresponds.

Example 2 Synthesis ofN⁴-benzoyl-1-{3′-O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}cytosine

First 2′-substitution, then 4′-substitution, then migration reaction:

All batches were carried out under an N₂ atmosphere.

N⁴-Benzoyl-1-(2′-O-benzoyl-β-D-ribopyranosyllcytosine 2

54.0 g (0.155 mol) of N⁴-benzoyl-1-(β-D-ribopyranosyl)cytosine 1 weredissolved in 830 ml of dimethylformamide (DMF) and 1.5 l of pyridine(both solvents dried and stored over molecular sieve 3 Å) with warmingto 124° C. 23.0 g (0.163 mol; 1.05 eq.) of BzCl, dissolved in 210 ml ofpyridine, were added dropwise at −58° to −63° C. in the course of 3.5 h.The batch was stirred overnight in a cooling bath. 90.3 g (1.5 mol; 10eq.) of n-propanol were stirred in and the batch was concentrated at 40°C. in a high vacuum. Pyridine residues were removed by twice adding 150ml of toluene and concentrating again. 124.3 g of residue were dissolvedin 500 ml of CH₂Cl₂, extracted twice by stirring with 300 ml ofhalf-concentrated NaHCO₃ solution each time, and the precipitated solidwas filtered off and dried: 60.7 g of residue. The CH₂Cl₂ phase wasconcentrated: 25.0 g. Separate chromatography on silica gel 60 (40×10cm) with gradients (AcOEt/isohexane, 4:1, then pure AcOEt, thenAcOET/MeOH, 19:1 to 2:1) yielded (TLC (silica gel, AcOET)):

16.8 g of 2′, 4′ dibenzoate (24%) R_(f) 0.5 12.4 g of 1 (23%) R_(f) 0.035.4 g of 2 (51%) R_(f) 0.14

N⁴-Benzoyl-1-{3′-O-benzoyl-4′-O-[(4,4′-dimethyoxytriphenyl)methyl]-β-D-ribopyranosyl}cystosine3

35.4 g (78 mmol) of 2 were dissolved in 390 ml of CH₂Cl₂ and 180 ml ofpyridine (both anhydrous) and 0.94 g (7.8 mmol; 0.1 eq.) of DMAP, 34.6ml (203 mmol; 2.6 eq.) of N-ethyldiisopropylamine and 33.1 g (98 mmol;1.25 eq.) of DMTCl were added and the mixture was stirred at RT. for 2h.

-   TLC (silica gel, AcOEt): R_(f) 0.6.-   CH₂Cl₂ was stripped off at 30° C., the residue was treated with 640    ml of pyridine, 9.37 g (78 mmol; 1.0 eq.) of DMAP, 32.5 ml (234    mmol; 3.0 eq.) of Et₃N, 21.7 g (156 mmol; 2.0 eq.) of p-nitrophenol    and 93.8 g (1.56 mol; 20 eq.) of n-propanol and stirred at 65° C.    for 42 h. The batch was concentrated in a high vacuum at 50° C.,    treated twice with 250 ml of toluene each time and concentrated. The    residue was taken up in 1 l of CH₂Cl₂, extracted three times by    stirring with 500 ml of dilute NaHCO₃ soln. each time, and the org.    phase was dried using Na₂SO₄ and concentrated: 92.5 g of residue.    Chromatography on silica gel 60 (50×10 cm) using gradients (methyl    tert-butyl ether/isohexane, 2:1 to 4:1, then methyl tert-butyl    ether/AcOEt, 1:4, then AcOEt/MeOH, 1:1 to 1:3) yielded 44.7 g of    product-containing fraction, which was recrystallized from 540 ml of    CH₂Cl₂/methyl tert-butyl ether, 1:5. The crystallizate was    recrystallized again from 300 ml of CH₂Cl₂/methyl tert-butyl ether,    1:1.-   3: TLC (silica gel, CHCl₃/i-PrOH 49:1): R_(f) 0.14.

The following was obtained: 30.0 g ofN⁴-benzoyl-1-{3′-O-benzoyl-4′-O[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}cytosine3 i.e. 51% yield based on 2. ¹H-NMR corresponds.

Example 3 Synthesis ofN⁶-benzoyl-9-(3′-O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}adenine

First 2′-substitution, then 4′-substitution, then migration reaction:

9-(β-D-Ribopyranosyl)adenine 2

68.37 g (100 mmol) ofN⁶-benzoyl-9-(2′,3′,4′-tri-O-benzoyl-β-D-ribopyranosyl)adenine 1 wasstirred overnight at RT. in 300 ml of NH₃-saturated MeOH and thecrystallizate was filtered off: 23.5 g (88%) of 2.

TLC (silica gel, AcOEt/MeOH 2:1): R_(f) 0.23/¹H-NMR (300 MHz, DMSO):3.56–3.78 (m, 3H, H(4′), H(5′)); 4.04 (m, 1H, H(3′)); 4.23 (ddd, J=2.5,8, 9.5 Hz, H(2′)), 4.89 (d, J=6 Hz, 1H, OH), 5.07 (d, J=7 Hz, 1H, OH),5.12 (d, J=4 Hz, 1H, OH), 5.63 (d, J=9.5 Hz, 1H, H(1′)), 7.22 (s, 2H,NH2), 8.14 (s, 1H, H(2)), 8.29 (s, 1H, H(8)).

¹³C-NMR (75 MHz, DMSQ): 65.0 (t, C(5′)); 66.6 (s, C(4′)), 68.1 (s,C(3′), 71.1 (s, C(2′)), 79.6 (s, C(1′)); 118.6 (C(5)); 139.5 (s, C(8)),149.9 (s, C(4)), 152.5 (s, C(2)), 155.8 (s, C(6)).

N⁶,N⁶-Dibenzoyl-9-(β-D-ribopyranosyl)adenine 3

16.8 g (62.9 mmol) of 2 were suspended in 500 ml of anhydrous pyridineunder an N₂ atmosphere and cooled to −4 to −10° C. 40 ml (199 mmol; 5eq.) of trimethylchlorosilane were added dropwise in the course of 20min and the mixture was stirred for 2.5 h with cooling.

-   36.5 ml (199 mmol; 5 eq.) of benzoyl chloride, dissolved in 73 ml of    pyridine, were added at −10 to −15° C. in the course of 25 min, and    stirred for 10 min with cooling and 2 h at RT. (TLC checking (silica    gel, AcOEt/heptane 1:1): R_(f) 0.5). The mixture was cooled again to    −10° C., 136 ml of H₂O (temp. max. +8° C.) were allowed to run in    and the mixture was stirred overnight at RT. After conversion was    complete, the solvent was stripped off and the residue was taken up    twice in 200 ml of toluene each time and evaporated again. The    mixture was treated with 500 ml each of Et₂O and H₂O, stirred    mechanically for 2 h, and the product which was only slightly    soluble in both phases was filtered off, washed with Et₂O and H₂O    and dried over P₂O₅ in a high vacuum: 23.8 g (80%) of 3.-   TLC (silica gel, AcOEt/MeOH 9:1): R_(f) 0.35.

¹H-NMR (300 MHz, DMSO): 3.60–3.80 (m, 3H, H(4′), H(5′)); 4.06 (bs, 1H,H(3′)); 4.30 (ddd, J=2.5, 8, 9.5 Hz, H(2′)), 4.93 (d, J=6 Hz, 1H, OH),5.20 (d, J=4 Hz, 1H, OH), 5.25 (d, J=4 Hz, 1H, OH), 5.77 (d, J=9.5 Hz,1H, H(1′)), 7.47 (m, 4H, Harom), 7.60 (m, 2H, Harom), 7.78 (m, 4H,Harom), 8.70 (s, 1H, H—C(2), 8.79 (s, 1H, H(8)).

¹³C-NMR (75 MHz, DMSO): 66.2 (t, C(5′)); 66.5 (s, C(4′)), 68.0 (s,C(3′)), 71.0 (s, C(2′)), 80.4 (s, C(1′)); 112.42 (C(5)); 126.9 (s,C(5′)), 126.9, 128.9, 133.3, 133.4 (arom. C), 146.0 (s, C(8)), 150.7 (s,C(4)), 151.8 (s, C(2)), 153.3 (s, C(6)) 172.0 (s, C═O)).

N⁶,N⁶-Dibenzoyl-9-(2′-O-benzoyl-β-D-ribopyranosyl)adenine 4

26.4 g (55,5 mmol) of 3 were dissolved in 550 ml of anhydrous CH₂Cl₂ and55 ml of pyridine (in each case stored over a molecular sieve) under anN₂ atmosphere, treated with 0.73 g (5.55 mmol; 0.1 eq.) of DMAP andcooled to −87 to −90° C. 8.58 g (61 mmol; 1.1 eq.) of BzCl in 14 ml ofpyridine were added dropwise in the course of 1 h and the mixture wasleft at −78° C. over a period of 60 h (week-end). The batch wasconcentrated, treated twice with 100 ml of toluene each time andevaporated in order to remove pyridine. Chromatography on silica gel 60(20×10 cm) using gradients (AcOEt/heptane, 1:1 to 9:1) yielded 23.2 g of4.

-   4: TLC (silica gel, AcOEt): R_(f) 0.34.

N⁶-Benzoyl-9-{3′-O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}adenine5

23.2 g (40 mmol) of 4 were dissolved in 160 ml of anhydrous CH₂Cl₂ andsubsequently treated with 14.9 g (56 mmol; 1.1 eq.) of DMTCl and 17.7 ml(104 mmol; 2.6 eq.) of N-ethyldiisopropylamine. After stirring at RT for2 h, a further 4.0 g (11.8 mmol; 0.3 eq.) of DMTCl were added and themixture was stirred for a further 40 min. The batch was concentrated ina Rotavapor at 350–520 mbar and 35° C.

-   TLC (silica gel, AcOEt/heptane 1:1): R_(f) 0.18.    The residue was dissolved in 260 ml of anhydrous pyridine and    subsequently treated with 51 ml (679 mmol; 17 eq.) of n-propanol,    16.6 ml (120 mmol; 3 eq.) of Et₃N, 11.1 g (80 mmol; 2 eq.) of    p-nitrophenol and 5.3 g (44 mmol; 1.1 eq.) of DMAP and stirred at    60–63° C. for 23 h. The batch then remained at RT. for 21 h. The    reaction mixture was concentrated in a Rotavapor. The residue was    treated twice with 200 ml of toluene each time and concentrated,    dissolved in CH₂Cl₂ and extracted three times with water.-   Chromatography on silica gel 60 (30×10 cm) using gradients    (AcOEt/heptane, 1:2 to 1:0; then AcOEt/MeOH, 1:0 to 9:1) yielded 13    g of 5.-   5: TLC (silica gel, AcOEt/heptane 4:1): R_(f) 0.2.    The following was obtained: 13 g of    N⁶-benzoyl-9-{3′-O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl-β-D-ribopyranosyl}adenine    5 i.e. 30% yield based on 3. ¹H-NMR corresponds. Time saving    compared with process known from the literature: 50%.

Example 4 Synthesis of9-[3′-O-benzoyl-4′-O-((4,4′-dimethoxytriphenyl)methyl)-β-D-ribopyranosyl]-2-O-allyl-2-N-isobutyroylguanine

First 3′-substitution, then 4′-substitution:

9-[3′-O-Benzoyl-β-D-ribopyranosyl]-2-O-allyl-2-N-isobutyroylguanine B

The G triol A (393 mg, 1.0 mmol) was dissolved in 4 ml of drydichloromethane. The solution was treated with trimethyl orthobenzoate(0.52 ml, 3.0 mmol) and camphorsulphonic acid (58 mg, 0.25 mmol) andstirred for 15 h at room temperature. The mixture was then cooled to 0°C. and treated with 2 ml of mixture of acetonitrile, water andtrifluoroacetic acid (50:5:1), which was precooled to 0° C. The mixturewas stirred for 10 min and the solvent was removed in vacuo. The residuewas purified by flash chromatography on silica gel (2.3×21 cm) usingdichloromethane/methanol 100:3. 25 mg (5%) of 4-O-benzoyl compound 139mg (28%) of mixed fractions and 205 mg (41%) of the desired 3-O-benzoylcompound B were obtained.

¹H-NMR (300 MHz, CDCl₃): 1.12, 1.14 (2d, J=7.0 Hz, 2×3H, NHCOCHMe ₂),2.78 (hep, J=7 Hz, 1H, NHCOCHMe ₂), 3.85 (dd, J=6.0, 11.0 Hz, 1H,H-5′_(eq)), 3.94 (app. T, J=11.0 Hz, 1H, H=5′_(ax)), 4.12 (ddd, J=2.5,6.0, 11.0 Hz, 1H, H-4′), 4.52 (dd, J=3.5, 9.5 hz, 1H, H-2′), 5.00 (dt,J=1.5, 6.0 Hz, 2H, All), 5.19 (dq, J=1.5, 10.0 Hz, 1H, All), 5.39 (dq,1.5, 16.5 Hz, 1H, All), 5.85 (bt, J=3.0 Hz, 1H, H-3′), 5.97 (d, J=9.5Hz, 1H, H-1′), 6.07 (ddd, J=6.0, 10.0, 16.5 Hz, 1H, All) 7.40–7.58 (m,3H. Bz), 8.10–8.16 (m, 2H, Bz), 8.28 (s, 1H, H-8).

9-[3′-O-Benzoyl-4′-O-((4,4′-dimethyloxytriphenyl)methyl)-β-D-ribopyranosyl]-2-O-allyl-2-N-isobutyroylguanineC

The diol B (101 mg, 0.2 mmol) was suspended in 3.2 ml of drydichloromethane. The suspension was treated with 171 μl (1.0 mmol) ofN-ethyldiisopropylamine, 320 μl (3.96 mmol) of pyridine and 102 mg (0.3mmol) of DMTCl and stirred at room temperature. After 24 h, a further102 mg (0.3 mmol) of DMTCl were added and the mixture was again stirredfor 24 h. It was then diluted with 30 ml of dichloromethane. Thesolution was washed with 20 ml of 10% strength aqueous citric acidsolution and 10 ml of saturated sodium bicarbonate solution, dried overMgSO₄ and concentrated in vacuo. The residue was purified by flashchromatography on silica gel (2.3×20 cm) using dichloromethane/methanol100:1. 39 mg of the known, desired product C (24%) were obtained.

Example 5 Synthesis of p-RNA Linker Systems

Three ways are described below which make possible the provision oflinkers which have an amino terminus, which can then be used for thelinkage of functional units:

5.1 Uracil-based Linker

on the basis of the modification of the 5-position of the uracil.

The preparation of hydroxyethyluracil 28 is possible on a large scaleaccording to a known method (J. D. Fissekis, A. Myles, G. B. Brown, J.Org. Chem. 1964, 29, 2670). g-Butyrolactone 25 was formylated withmethyl formate, the sodium salt 26 was reacted to give the ureaderivative 27 and this was cyclized to give the hydroxyethyluracil 28(Scheme 4).

Hydroxyethyluracil 28 was mesylated with methanesulphonyl chloride inpyridine to give 29 (J. D. Fissekis, F. Sweet, J. Org. Chem. 1973, 38,264).

The following stages have been newly invented: using sodium azide inDMF, 29 was reacted to give the azide 30 and this was reduced withtriphenylphosphine in pyridine to give the aminoethyluracil 31. Theamino function in 31 was finally protected withN-ethyoxycarbonylphtalimide (Scheme 5). Nucleosidation of ribosetetrabenzoate 33 with N-phtaloylaminoethyluracil 32 yielded the ribosetribenzoate 34 in good yields. The anomeric centre of the pyranose ring,as can be clearly seen from the coupling constant between H—C(1′) andH—C(2′) of J=9.5 Hz, has the β configuration. Subsequent removal of thebenzoate protective groups with NaOMe in MeOH yielded the linker triol35. 35 was reacted with benzoyl chloride at −78° C. inpyridine/dichloromethane 1:10 in the presence of DMAP. In this process,in addition to the desired 2′-benzoate 36 (64%), 2′,4′-dibenzoylatedproduct (22%) was obtained, which was collected and converted again intothe triol 35 analogously to the methanolysis of 34 to, 35. The2′-benzoate 36 was tritylated in the 4′-position in yields. of greaterthan 90% using dimethoxytrityl chloride in the presence of Hünig's basein dichloromethane. The rearrangement of 4′-DMT-2′-benzoate 37 to the4′-DMT-3′-benzoate 38 was carried out in the presence of DMAP,p-nitrophenol and Hünig's base in n-propanol/pyridine 5:2. Afterchromatography, 38 is obtained. 4′-DMT-3′-benzoate 38 was finallyreacted with ClP(OAll)N(iPr)₂ in the presence of Hünig's base to givethe phosphoramidite 39 (Scheme 6). This can be employed for theautomated oligonucleotide synthesis without changing the synthesisprotocol.

Procedure Synthesis of a Uracil Linker Unit 5-(2-Azidoethyl)uracil (30)

1. Procedure

26.0 g (0.11 mol) of 29 were dissolved in 250 ml of DMF in a 500 mlthree-necked flask equipped with an internal thermometer and refluxcondenser and the mixture was treated with 10.8 g (0.166 mol) of sodiumazide. The suspension was subsequently stirred at 60° C. for 4 hours(TLC checking, CHCl₃:MeOH 9:1). The DMF was distilled off and theresidue was stirred with 150 ml of water. The solid was filtered off,washed with about 50 ml of water and dried overnight over phosphoruspentoxide in vacuo in a desiccator. 14.2 g (71%) of 30 were obtained inthe form of a colourless solid of m.p. 230–235° C. (with dec.).

2. Analytical Data 5-(2-Azidoethyl)uracil (30)

M.p. 230–235° C. with decomp. TLC: CHCl₃/MeOH 9:1, R_(f) 0.48 UV (MeOH):λ_(max) 263.0 (7910). IR (KBr): 3209s, 3038s, 2139s, 1741s, 1671s,1452m, 1245m, 1210m ¹H-NMR (300 MHz, 2.46 (t, 2H, J(CH₂CH₂N, d₆-DMSO):CH₂CH₂N) = 7.0, CH₂CH₂N); 3.40 (t, 2H, J(CH₂CH₂N, CH₂CH₂N) = 7.0CH₂CH₂N); 7.36 (s, H—C(6)); 11.00 (br. s, 2H, H—N(1), H—N(3). MS (ESI⁺):180.0 [M + H]. .

5-(2-Aminoethyl)uracil (31)

1. Procedure

14.2 g (78.0 mmol) of 30 were suspended in 175 ml of pyridine in a 250ml three-necked flask equipped with an internal thermometer and refluxcondenser and the mixture was treated with 61.4 g (234 mmol) oftriphenylphosphine²⁾. It was heated at 60° C. for 5 hours and stirredovernight at room temp. (TLC checking, CHCl₃/MeOH 5:1). 40 ml of 25%strength ammonia solution were added to the suspension, which thenclarified. The solvents were removed in vacuo in a rotary evaporator.The residue was stirred at room temp. for 30 min in 200 ml ofCH₂Cl₂/MeOH 1:1, and the precipitate was filtered off and washed withCH₂Cl₂. After drying in vacuo in a desiccator over phosphorus pentoxide,10.0 g (85%) of 31 of m.p. 214–220° C. were obtained.

2. Analytical Data 5-(2-Aminoethyl)uracil (31)

M.p. 214–220° C. with evolution of gas, presintering. TLC:CHCl₃/MeOH/HOAc/H₂O 85:20:10:2, R_(f) 0.07 UV (MeOH): λ_(max) 263.0(6400). IR (KBr): 3430m, 3109s, 1628s, 1474m, 1394s, 1270s, 1176w,1103m, 1021m, 966m, 908m, 838m. ¹H-NMR (300 MHz, 2.21 (t, 2H, J(CH₂CH₂N,d₆-DMSO): CH₂CH₂N) = 6.8, CH₂CH₂N); 2.59 (t, 2H, J(CH₂CH₂N, CH₂CH₂N) =6.8 CH₂CH₂N); 5.90 (v. br. s, 4H, H—N(1), H—N(3), NH₂); 7.19 (s,H—C(6)). MS (ESI⁻): 153.9 [M − H].

5-(2-Phtalimidoethyl)uracil (32)

1. Procedure

9.6 g (61.8 mmol) of 31 were suspended in 100 ml of water in a 250 mlround-bottomed flask and treated with 6.64 g (62.6 mmol) of Na₂CO₃.After stirring at room temp. for 15 min, 14.3 g (65 mmol) ofN-ethoxycarbonylphtalimide were added in portions and the mixture wasstirred for three hours at room temp. (TLC checking, CHCl₃/MeOH 5:1).The now viscous, white suspension was carefully¹⁾ adjusted to pH 4 usingconc. hydrochloric acid and the white precipitate was filtered off.After washing with water, the solid was dried over phosphorus pentoxidein a desiccator in vacuo. This yielded 16.0 g (91%) of 32 of m.p.324–327° C.

2. Analytical Data 5-(2-Phtalimidoethyl)uracil (32)

M.p. 324–327° C. with decomp. TLC: CHCl₃/MeOH 5:1, R_(f) 0.51 UV (MeOH):λ_(max) 263.0 (5825); λ 298.0 (sh., 1380). IR (KBr): 3446m, 3216m,1772m, 1721s, 1707s, 1670s, 1390m. ¹H-NMR (300 MHz, 2.49 (t, 2H,J(CH₂CH₂N, d₆-DMSO): CH₂CH₂N) = 6.0, CH₂CH₂N); 3.71 (t, 2H, J(CH₂CH₂N,CH₂CH₂N) = 6.0 CH₂CH₂N); 7.24 (s, H—C(6)); 7.84 (m_(c), 4H, NPht); 10.76(br, s, H—N(1), H—N(3)). MS (ESI⁻): 284.0 [M − H].

1-(2,3,4-Tri-O-benzoyl-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil(34)

1. Procedure

7.00 g (24 mmol) of 32 and 13.6 g (24 mmol) of 33 were suspended in 120ml of acetonitrile in a 250 ml three-necked flask, equipped with anargon lead-in, internal thermometer and septum. Firstly 12.2 ml (50mmol) of BSA and, after stirring for 30 min, a further 7 ml (28 mmol) ofBSA were then added by means of syringe. After heating to 40° C. for ashort time, the reaction mixture clarified. 13 ml (72 mmol) of TMSOTfwere added by means of syringe at room temp. After one hour, no productformation was yet observed (TLC checking, AcOEt/n-heptane 1:1). Afurther 13 ml (72 mmol) of TMSOTf were therefore added. Subsequently,the reaction mixture was heated to 50° C. After stirring at 50° C. for2.5 h (TLC checking), the mixture was cooled to RT., [lacuna] onto anice-cold mixture of 250 ml of AcOEt and 190 ml of satd. NaHCO₃ solutionand intensively extracted by stirring for 10 min. It was again washedwith 100 ml of NaHCO₃ solution and the aqueous phases were againextracted with 100 ml of AcOEt. The dil. org. phases were dried usingMgSO₄ and the solvents were removed in vacuo in a rotary evaporator.After drying in an oil pump vacuum, 20.9 g of crude product wereobtained. Chromatography on silica gel (h=25 cm, Φ=5 cm, AcOEt/n-heptane1:1) yielded a TLC-uniform, foamy product, which was digested usingEt₂O. Fitration and drying in an oil pump vacuum afforded 15 g (86%) of34.

2. Analytical Data1-(2,3,4-Tri-O-benzoyl-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil(34)

M.p. 124° C. (sintering) TLC: AcOEt/n-heptane 1:1, Rf 0.09. UV (MeOH):λ_(max) 263.0 (11085): λ 299.0 (sh., 1530) IR (KBr): 3238w, 3067w,1772m, 1710s, 1452m, 1395m, 1266s, 1110s, 1070m, 1026m. ¹H-NMR (300 MHz,2.79 (m_(c), 2H, CH₂CH₂N); 3.96 CDCl₃): (m_(c), 2H, CH₂CH₂N); 4.06 (dd,J(H_(eq)-C(5′), H_(ax)-C(5′)) = 11.0, J(H_(eq)-C(5′), H—C(4′)) = 6.0,H_(eq)-C(5′)); 4.12 (t, J(H_(ax)-C(5′), H_(eq)-C(5′)) = J(H_(ax)-C(5′),H—C(4′)) = 11.0, H_(ax)-C(5′)); 5.39 (dd, J(H—C(2′), H— C(1′)) = 9.5,J(H—C(2′), H—C(3′)) = 2.9 H—C(2′)); 5.46 (ddd, J(H—C(4′), H_(ax)- C(5′))= 11.0, J(H—C(4′), H_(eq)-C(5′)) = 6.0, J(H—C(4′), H—C(3′)) = 2.9, H—C(4′)); 6.26 (ψt, J ≈ 2.6, H—C(3′)); 6.36 (d, J(H—C(1′), H—C(2′)) = 9.5,H— C(1′)); 7.24–7.40, 7.44–7.56, 7.61–7.66, 7.72–7.80, 7.84–7.90,8.06–8.13 (6 m, 16H, 3 Ph, H—C(6)); 7.70, 7.82 (2 m_(c), 4H, NPht); 8.37(s, H—N(3)). ¹³C-NMR (75 MHz, 21.19 (CH₂CH₂N); 36.33 CDCl₃): (CH₂CH₂N);64.07 (C(5′)); 66.81, 68.22 (C(4′), C(2′)); 69.29 (C(3′)); 78.59(C(1′)); 112.42 (C(5)); 123.31, 132.05, 133.89 (6C, Pht); 128.33,128.47, 128.47, 128.83, 128,86, 129.31, 129.83, 129.83, 129.94, 133.55,133.62, 133.69 (18C, 3 Ph); 135.87 (C(6)); 150.39, 162.78 (C(4));164.64, 165.01, 165.41 (3C, O₂CPh); 168.43 (2C, CO—Pht). MS (ESI⁺):730.2 [M + H].

Anal.: calc. for C₄₀H₃₁N₃O₁₁ (729.70): C, 65.84, H, 4.28, N, 5.76;found: C, 65.63, H, 4.46, N, 5.53.

5-(2-Phtalimidoethyl)-1-(β-D-ribopyranosyl)uracil (35)

1. Procedure

15 g (20 mmol) of 34 were dissolved in 500 ml of MeOH in a 1 lround-bottomed flask, treated with 324 mg (6 mmol) of NaOMe and stirredat room-temp. overnight with exclusion of water (TLC checking,AcOEt/n-heptane 1:1). Amberlite IR-120 was added to the resultingsuspension until the pH was <7. The solid was dissolved in the presenceof heat, filtered off hot from the ion exchanger and washed with MeOH.After removing the solvent, the residue was co-evaporated twice using150 ml of water each time. This yielded 9 g of crude product, which washeated under reflux in 90 ml of MeOH for 10 min. After cooling to roomtemp., the mixture was treated with 60 ml of Et₂O and stored overnightat 4° C. Filtration, washing with Et₂O and drying in an oil pump vacuumyielded 7.8 g (93%) of 35.

2. Analytical Data 5-(2-Phtalimidoethyl)-1-(β-D-ribopyranosyl)uracil(35)

M.p. 137° C. (sintering) TLC: CHCl₃/MeOH 5:1, R_(f) 0.21. UV (MeOH):λ_(max) 263.0 (8575): λ 299.0 (sh., 1545). IR (KBr): 3458s, 1772w,1706s, 1400m, 1364m, 1304m, 1045m. ¹H-NMR (300 MHz, 2.55 (m_(c), 2H,d₆-DMSO + 2 Tr. D₂O: CH₂CH₂N); 3.28–3.61 (m, 4H, H—C(2′), H— C(4′),H_(eq)-C(5′), H_(ax)-C(5′)); 3.73 (m_(c), 2H, CH₂CH₂N); 3.93 (m,H—C(3′)); 5.50 (d, J(H—C(1′), H—C(2′)) = 9.3, H—C(1′)); 7.41 (s,H—C(6)); 7.84 (s, 4H, NPht). ¹³C-NMR (75 MHz, 25.63 (CH₂CH₂N); 36.62d₆-DMSO): (CH₂CH₂N); 64.95 (C(5′)); 66.29 (C(4′)); 67.37 (C(2′)); 71.12(C(3′)); 79.34 (C(1′)); 110.39 (C(5)); 122.85, 131.54, 134.08 (6C, Pht);137.92 (C(6)); 150.84 (C(2)); 163.18 (C(4)); 167.74 (2C, CO— Pht). MS(ESI⁻): 416.1 [M − H].

1-(2′-O-Benzoyl-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil

10.6 g (0.025 mmol) of 5-(2-phtalimidoethyl)-1-(β-D-ribopyranosyl)uracilwere dissolved in 20 ml of pyridine in a heated and argon-flushed 1 lfour-necked flask and mixed with 200 ml of dichloromethane. The mixturewas cooled to −70° C., 3.82 ml (0.033 mmol) of benzoyl chloride in 5 mlof pyridine and 20 ml of dichloromethane were slowly added dropwise withcooling and the mixture was stirred at −70° C. for 35 min. The reactionmixture was poured onto 600 ml of cooled ammonium chloride solution andthe aqueous phase was extracted with ethyl acetate. The combined organicphases were washed with water, dried and concentrated to dryness invacuo. Chromatography on silica gel (ethyl acetate/heptane 1:1) yielded7.9 g (60%) of1-(2′-O-benzoyl-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil.

-   TLC: R_(f) 0.24 (ethyl acetate/heptane 4:1)-   ¹H-NMR (300 Mhz, d₆-DMSO) 2.67 (m_(e) 2H, CH₂CH₂N); 3.66–3.98 (m,    5H, H—C(4′), H_(eq)—C(5′), H_(ax)—C(5′), CH₂CH₂N) 4.51 (t, 1H,    H—C(3′)); 4.98 (dd, 1H, H—C(2′)); 6.12 (d, 1H, H—C(1′)); 7.19 (s,    1H, H—C(6)); 7.29–7.92 (m, 9H, OBz, NPht).

1-(2-O-Benzoyl-4-O-(4,4′-dimethoxytrityl)-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil

5.6 g (10.73 mmol) of1-(2-O-benzoyl-β-D-ribopyranosyl-5-(2-phtalimidoethyl)uracil weredissolved in 60 ml of dichloromethane, treated with 4.72 g (13.95 mmol)of 4,4′-dimethoxytrityl chloride and 2.4 ml (13.95 mmol) ofN-ethyldiisopropylamine and stirred at RT for 20 min. The reactionmixture was diluted with 100 ml of dichloromethane, washed with sodiumhydrogencarbonate solution and 20% citric acid solution, dried andconcentrated to dryness in vacuo. Chromatography on silica gel (ethylacetate/heptane 1:1+2% triethylamine) yielded 7.7 g (87%) of1-(2-O-benzoyl-4-O-(4,4′-dimethoxytrityl)-β-ribopyransoyl)-5-(2-phtalimidoethyl)uracil.

-   TLC: R_(f) 0.53 (ethyl acetate/heptane 1:1+2% triethylamine).

¹H-NMR (300 MHz, CDCl₃): 2.64 (m_(c), 2H, CH₂CH₂N) ; 3.12 (m_(c), 1H,H—C(4′)); 3.59–3.63 and 3.72–3.92 (m, 5H, H—C(3′), H_(eq)—C(5′),H_(ax)—C(5′), CH₂CH₂N); 3.81 and 3.82 (s, 6H, CH₃O); 4.70 (dd, 1H,H—C(2′)); 6.09 (d, 1H, H—C(1′)), 7.05 (s, 1H, H—C(6)); 6.84–7.90 (m,22H, ODmt, OBz, NPht).

1-(3-O-Benzoyl-4-O-(4,4′-dimethoxytrityl)-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil

3 g (3.63 mmol) of1-(2-O-Benzoyl-4-O-(4,4′-dimethoxytrityl)-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil,1 g (7.26 mmol) of 4-nitrophenol, 0.44 g (3.63 mmol) of4-(dimethylamino)pyridine and 3.75 ml (21.78 mmol) ofN-ethyldiisopropylamine were dissolved in 5.6 ml of isopropanol and 25ml of pyridine, heated to 65° C. and stirred at 65° C. for 3 days. Thesolution was concentrated to dryness in vacuo and the residue wasdissolved in 150 ml of dichloromethane. After washing with 20% citricacid solution and sodium hydrogencarbonate solution, the solution wasdried over magnesium sulphate. Chromatography on silica gel (ethylacetate/dichloromethane/isohexane 2:1:1) yielded 2.27 g (76%) of1-(3-O-benzoyl-4-O-(4,4′-dimethoxytrityl)-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracil.

-   TLC: R_(f) 0.27 (ethyl acetate/isohexane 2:1+1% triethylamine).

¹H-NMR (300 MHz, CDCl₃): 2.39 (m_(c), 2H, CH₂CH₂N); 2.53 (m_(c), 1H,H_(eq)—C(5′)); 3.30 (dd, 1H, H—C(2′)); 3.55 (m_(c), 1H, H_(ax)—C(5′));3.69 (m_(c), 2H, CH₂CH₂N); 3.78 and 3.79 (s, 6H, CH₃O); 3.79–3.87 (m,1H, H—C (4′)); 5.74 (d, 1H, H—C(1′)); 5.77 (m_(c), 1H, H—C(3′)); 6.92(s, 1H, H—C(6)); 6.74–8.20 (m, 22H, ODmt, OBz, NPht).

1-{2′-O-[(Allyloxy)(diisopropylamino)phosphino]-3′O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}-5-(2-phtalimidoethyl)uracil

88 mg (0.11 mmol) of1-(3-O-benzoyl-4-O-(4,4′-dimethoxytrityl)-β-D-ribopyranosyl)-5-(2-phtalimidoethyl)uracilwere dissolved in 5 ml of dichloromethane, treated with 75 μl (0.44mmol) of N-ethyldiisopropylamine and 70 μl (0.3 mmol) ofallyloxychloro(diisopropylamino)phosphine and stirred for 3 h at roomtemperature. After addition of a further 35 μl (0.15 mmol) ofallyloxychloro(diisopropylamino)phosphine to complete the reaction, itwas stirred for a further 1 h at room temperature and the reactionmixture was concentrated in vacuo. Chromatography on silica gel (ethylacetate/heptane: gradient 1:2 to 1:1 to 2:1, in each case with 2%triethylamine) yielded 85 mg (76%) of1-{2′-O-[(allyloxy)(diisopropylamino)phosphino]-3′O-benzoyl-4′-O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}-5-(2-phtalimidoethyl)uracil.

-   TLC: R_(f) 0.36 (ethyl acetate/heptane 2:1).

¹H-NMR (,CDCl₃, 300 MHz,): selected characteristic positions: 2.28, 2.52(2 dd, J=5.0, 11.0 Hz, 2H, 2H-5′), 3.79, 3.78 (app. 2 s, 12H, OMe), 6.14(1 bs, 1H, H-3′).

³¹P-NMR (CDCl₃): 149.8, 150.6

5.2 Indole-based Linker

N-phthaloyltryptamine is obtained from phthalic anhydride and tryptamineas described (Kuehne et al J. Org. Chem. 43, 13, 1978, 2733–2735). Thisis reduced with borane-THF to give the indoline (analogously to A.Giannis, et al., Angew. Chem. 1989, 101, 220).

The 3-substituted indoline is first reacted with ribose to give thenucleoside triol and then with acetic anhydride to give the triacetate.The mixture is oxidized with 2,3-dichloro-5,6-dicyanoparaquinone and theacetates are cleaved with sodium methoxide, benzoylated selectively inthe 2′-position, DM-tritylated selectively in the 4′-position, and themigration reaction is carried out to give the 3′-benzoate. The formationof the phosphoramidite is carried out in the customary manner. This canbe employed for the automated oligbnucleotide synthesis withoutalteration of the synthesis protocols.

Procedure 3-(N-Phthaloyl-2-aminoethyl)indoline

51.4 g (177 mmol) of phthaloyltryptamine A were dissolved in 354 ml of1M borane-THF solution (2 eq.) under a nitrogen atmosphere and cooled to0° C. 354 ml of trifluoroacetic acid were slowly added dropwise at 0° C.(caution: evolution of gas) and the mixture was stirred for 30 min. (TLCchecking: EtOAc). 17.3 ml of water were then added, and the mixture wasstirred for 10 min and concentrated in vacuo. The residue was dissolvedin 10% strength NaOH solution/dichloromethane, and the organic phase wasseparated off, dried over NaSO₄, filtered and concentrated in vacuo. Theresidue [50.9 g] was recrystallized from hot ethanol (3 l). 41.4 g of Bwere obtained, m.p. 161–162° C. The mother liquor was concentrated invacuo and the residue was again recrystallized from ethanol. A further3.2 g of B were obtained, m.p. 158–159° C.

Total yield: 44.6 g (153 mmol) of B, i.e. 86%.

-   ¹H-NMR (CDCl₃, 300 MHz): 1.85–2.00, 2.14–2.28 (2 m, 2×1H, CH    ₂CH₂NPhth); 2.70 (bs, 1H, NH), 3.24–3.38, 3.66–3.86 (2 m, 5H, CH₂CH    ₂NPhth, H-2a, H-2b, H-3), 6.62 (d, J=8.0 Hz, 1H, H-7), 6.66–6.72 (m,    1H, H-5), 6.99 (app t, J=7.5 Hz, 1H, H-6), 7.14 (d, J=8.0 Hz, 1H,    H-4), 7.64–7.74, 7.78–7.86 (2 m, 2×2H, Phth).

¹³C-NMR (CDCl₃, 75 MHz): 32.70, 36.10 (2 t, C-2, CH₂CH₂NPhth), 39.62 (d,C-3), 53.04 (t, CH₂NPhth), 109.65 (d, C-7), 118.74 (d, C-5), 123.25 (d,Phth), 123.92, 127.72 (2 d, C-4, C-6), 131.81 (s, C-3a), 132.14 (s,Phth), 133.99 (d, Phth), 151.26 (s, C-7a), 168.38 (s, C═O).

Calc.: C: 73.96, H: 5.52, N: 9.58; found: C: 73.89, H: 5.57, N: 9.55.

MS (ES⁺): 293 (MH⁺, 100%)

3-(N-Phthaloyl-2-aminoethyl)-1-(2′,3′,4′-tri-O-acetyl-β-D-ribopyranosyl)indole

45.2 g (155 mmol) of A and 23.2 g (155 mmol; 1.0 eq.) of D-ribose weresuspended in 750 ml of dry ethanol and heated to reflux for 4 h under anitrogen atmosphere (TLC checking: CH₂Cl₂/MeOH 10:1). After cooling toRT, the mixture was concentrated in vacuo. The residue was dissolved in300 ml of pyridine and treated with 155 ml of acetic anhydride withice-cooling. After 15 min., the ice bath was removed and the mixture wasstirred at RT for 18 h (TLC checking: EtOAc/isohexane 1:1). Thissolution was concentrated in vacuo and co-evaporated three times with300 ml of toluene each time. The oil obtained was dissolved in 900 ml ofdichloromethane and treated with 38.8 g (171 mmol; 1.1 eq.) of2,3-dichloro-5,6-dicyanoparaquinone with ice-cooling. After 15 min., theice bath was removed and the mixture was stirred at RT for 1.5 h (TLCchecking: EtOAc/isohexane 1:1). The deposited precipitate was filteredoff with suction and washed with dichloromethane and discarded. Thefiltrate was washed with 600 ml of satd. NaHCO₃ solution. Theprecipitate deposited in the course of this was again filtered off withsuction and washed with dichloromethane and discarded. The combinedorganic extracts were dried over Na₂SO₄ and concentrated in vacuo. Theresidue (90.9 g) was purified by flash chromatography on silica gel 60(10×25 cm; EtOAc/isohexane 2:3).

The following were obtained: 21.5 g of pure B and 46.83 g of mixedfractions, which after fresh chromatography yielded a further 20.4 g ofpure B.

Total yield: 41.9 g (76 mmol) of B, i.e. 49%.

-   ¹H-NMR (CDCl₃, 300 MHz): 1.64, 1.98, 2.19 (3 s, 3×3H, Ac), 3.06 (t,    J=8.0 Hz, 2H, CH ₂CH₂NPhth), 3.81–4.00 (m, 4H, H-5′ax, H-5′eq, CH    ₂NPhth), 5.13 (ddd, J=2.5, 6.0, 10.5 Hz, 1H, H-4′), 5.36(dd, J=3.5,    9.5 Hz, 1H, H-2′), 5.71(d, J=9.5 Hz, 1H, H-1′), 5.74(app t, J=3.0    Hz, 1H, H-3′), 7.02(s, 1H, H-2), 7.04–7.10, 7.13–7.19 (2 m, 2×1H,    H-5, H-6), 7.33 (d, J=8.0 Hz, 1H, H-7), 7.58–7.66, 7.72–7.80(2 m,    5H, Phth, H-4).-   ¹³C-NMR (CDCl₃, 75 MHz): 20.23, 20.65, 20.87 (3 q, Ac), 24.41, 38.28    (2 t, CH₂CH₂), 63.53 (t, C-5′), 66.24, 68.00, 68.64 (3 d, C-2′,    C-3′, C-4′), 80.33 (d, C-1′), 109.79 (d, C-7), 113.95 (s, C-3),    119.33, 120.39, 122.04, 122.47 (4 d, C-4, C-5, C-6, C-7), 123.18 (d,    Phth), 128.70, 132.17 (2 s, C-3a, Phth), 133.87 (d, Phth), 136.78    (s, C-7a), 168.243, 168.77, 169.44, 169.87 (4 s, C═O).

Calc.: C: 63.50, H: 5.15, N: 5.11; found: C: 63.48, H: 5.16, N: 5.05.

MS (ES⁺): 566 (M+NH₄ ⁺, 82%), 549 (MH⁺, 74%), 114 (100%).

3-(N-Phthaloyl-2-aminoethyl)-1-β-D-ribo-pyranosyl-indol

44.1 g (80 mmol) of A were dissolved in 400 ml of anhydrous methanolunder a nitrogen atmosphere. The mixture was treated with 4.0 ml of 30%strength sodium methoxide solution with ice-cooling and then stirred for18 h at RT. The deposited precipitate was filtered off with suction andwashed with cold ethanol. The filtrate was concentrated in vacuo. Theresidue was taken up in dichloromethane. This solution was washed withsatd. NaHCO₃ solution, dried over Na₂SO₄ and concentrated in vacuo. Theresidue obtained was recrystallized from hot ethanol together with theprecipitate deposited from the reaction solution. 22.6 g of B wereobtained, m.p. 196–198° C. The mother liquor was concentrated in vacuoand the residue was again recrystallized from ethanol. A further 9.2 gof B were obtained, m.p. 188–194° C.

Total yield: 25.8 g of B, i.e. 76%.

¹H=NMR (MeOD, 300 MHz): 3.09 (app. t, J=7.0 Hz, 2H, CH ₂CH₂NPhth),3.64–3.98 (m, 5H, H-4′, H-5′ax, H-5′eq, CH ₂NPhth), 4.05 (dd, J=3.5, 9.5Hz, 1H, H-2′), 4.22 (app t, J=3.0 Hz, 1H, H-3′), 5.65 (d, J=9.5 Hz, 1H,H-1′), 6.95–7.05, 7.09–7.16 (2 m, 2×1H, H-5, H-6), 7.25 (s, 1H, H-2),7.44 (d, J=8.0 Hz, 1H, H-7), 7.60 (d, J=8.0 Hz, 1H, H-4), 7.74–7.84 (m,4H, Phth).

¹³C-NMR (d₆-DMSO, 75 MHz): 23.87, 37.79 (2 t, CH₂ CH₂NPhth), 64.82 (t,C-5′), 66.74 (d, C-4′), 68.41 (d, C-2′), 71.42 (d, C-3′), 81.37 (d,C-1′), 110.42 (d, C-7), 111.05 (s, C-3), 118.17, 119.21, 121.36, 122.92,123.80 (5 d, C-2, C-4, C-5, C-6, NPhth), 127.86, 131.59 (2 s, C-3a,Phth), 134.27 (d, Phth), 136.62 (s, C-7a), 167.72 (s, C═O).

MS(ES⁻): 457 (M+OH⁻+H₂O, 49%), 439 (M+OH⁻, 100%), 421 (M−H⁺, 28%)

1-(2′-O-Benzoyl-β-D-ribopyranosyl)-3-(N-phthaloyl-2-aminoethyl)indole

10.6 g (25 mmol) of A was taken up in 250 ml of dry dichloromethaneunder a nitrogen atmosphere. The mixture was treated with 305 mg of DMAP(2.5 mmol) and 20 ml of pyridine. It was heated until everything was insolution and then cooled to −78° C. 3.35 ml of benzoyl chloride (28.8mmol) dissolved in 8 ml of dichloromethane were now added dropwise inthe course of 15 min. TLC checking (EtOAc/hexane 3:1) after a further 30min indicated complete reaction. After 45 min, the cold solution wasadded directly to 200 ml of satd. NH₄Cl solution through a folded filterand the filter residue was washed with dichloromethane. The organicphase was washed once with water, dried over MgSO₄ and concentrated. Theresidue was co-evaporated twice with toluene and purified by flashchromatography on 10×20 cm silica gel using EtOAc/hexane 3:1. 8.1 g of B(64%) were obtained.

¹H-NMR (CDCl₃, 300 MHz): 2.45, 2.70 (2 bs, 2×1H, OH), 3.04 (t, J=8.0 Hz,2H, CH ₂CH₂NPhth), 3.80–4.20 (m, 5H, H-4′, H-5′ax, H-5′eq, CH ₂NPhth),4.63 (bs, 1H, H-3′), 5.46 (dd, J=3.5, 9.5 Hz, 1H, H-2′), 6.03 (d, J=9.5Hz, 1H, H-1′), 7.08–7.31 (m, 5H, H-2, H-5, H-6, Bz-m-H), 7.41–7.48 (m,1H, H-Bz-p-H), 7.50 (d, J=8.0 Hz, 1H, H-7), 7.64–7.79 (m, 7H, Phth, H-4,Bz-o-H).

¹³C-NMR (d₆-DMSO, 75 MHz): 24.40, 38.22 (2 t, CH₂ CH₂NPhth), 65.95 (t,C-5′), 66.65 (d, C-4′), 69.55 (d, C-3′), 71.87 (d, C-2′), 79.57 (d,C-1′), 109.96 (d, C-7), 113.70 (s, C-3), 119.21, 120.21, 122.11, 122.41,123.14, (5 d, C-2, C-4, C-5, C-6, NPhth), 128.28 (d, Bz), 128.58,128.59, (2 s, C-3a, Bz), 129.62 (d, Phth), 132.05 (s, Phth), 133.81(Bz), 136.97 (s, C-7a), 165.12, 168.29 (2 s, C═O).

MS (ES⁻): 525 (M−H⁺, 12%), 421 (M-PhCO⁺, 23%), 107 (100%).

1-{3′-O-Benzoyl-4′O-[(4,4′-dimethoxytriphenyl)methyl-β-D-ribopyranosyl}-3-(N-phthaloyl-2-aminoethyl)indole

8.9 g (16.9 mmol) of A was suspended in 135 ml of dry dichloromethaneunder a nitrogen atmosphere. The mixture was treated with 206 mg of DMAP(1.68 mmol), 5.8 ml of N-ethyldiisopropylamine (33.7 mmol) and about 12ml of pyridine (until solution was complete). It was now treated with 34g of molecular sieve 4 Å and stirred for 30 min. After cooling to 0° C.,it was treated with 11.4 g of DMTCl (33.7 mmol) and stirred for 75 minafter removing the cooling bath. A further 1.94 g (0.34 eq) and, after afurther 40 min, 1.14 g (0.2 eq) and, after a further 65 min, 1.14 g ofDMTCl (0.2 eq) were then added. After 4.25 h the reaction was complete.The mixture was then treated with 25.3 ml of n-propanol (20 eq), stirredfor a further 30 min and then concentrated cautiously (foam formation).The residue was dissolved in 100 ml of pyridine. It was treated with1.85 g of DMAP (15.1 mmol; 0.9 eq), 13.05 ml of N-ethyldiisopropylamine(101 mmol; 6.0 eq), 71 ml of n-propanol (940 mmol; 56 eq) and 3.74 g ofp-nitrophenol (26.9 mmol; 1.6 eq). This mixture was stirred undernitrogen for 96 h at 75–80° C. After cooling to room temperature, themixture was filtered through Celite and concentrated. The residue waspurified by flash chromatography on 9×17 cm silica gel usingtoluene/diethyl ether/triethylamine 90:10:1. The product-containingfractions (9.25 g) were first recrystallized from EtOAc and thenreprecipitated from toluene/methanol. 5.86 g of B (42%) were obtained.

¹H-NMR (CDCl₃, 300 MHz): 2.64 (bs, 1H, OH), 2.68 (dd, J=5.0, 11.5 Hz,1H, H-5′eq), 2.94 (dd, J=7.5, 16.0 Hz, 1H, CH ₂CH₂NPhth), 3.03 (dd,J=8.0, 16.0 Hz, 1H, CH ₂CH₂NPhth), 3.67–3.74 (m, 1H, H-5′ax), 3.69, 3.70(2 s, 2×3H, OMe), 3.85 (t, J=7.5 Hz, 2H, CH₂CH ₂NPhth), 3.94 (ddd,J=3.0, 5.0, 10.5 Hz, 1H, H-4′), 4.03 (dd, J=3.5, 9.0 Hz, 1H, H-2′), 5.51(d, J=9.0 Hz, 1H, H-1′), 5.86 (bs, 1H, H-3′), 6.68–7.66 (m, 25H),8.19–8.30 (m, 2H).

¹³C-NMR (CDCl₃, 75 MHz): 24.16, 38.80 (2 t, CH₂ CH₂NPhth), 55.25, 55.26(2 q, Ome), 65.58 (t, C-5′), 68.29, 69.19, 73.83 (3 d, C-2′, C-3′,C-4′), 83.03 (d, C-1′), 87.31 (CAr₃)110.03 (d, C-7), 113.37, 113.47 (2d), 113.53 (s, C-3), 118.95, 120.20, 122.28, 122.31, 123.10, 127.07,128.02, 128.08, 128.68 (9 d), 128.74 (s), 130.02, 130.19, 130.22 (3 d),130.37, 131.95 (2 s), 133.40, 133.83 (2 d), 135.98, 136.14, 136.56,145.12, 158.82, 166.76, 168.52 (7 s, C-7a, 2 COMe, 2 C═O).

1-{2′O-(Allyloxy)(diisopropylamino)phosphino)-3′-O-benzoyl-4′O-[(4,4′-dimethoxytriphenyl)methyl]-β-D-ribopyranosyl}-3-(N-phthaloyl-2-aminoethyl)indole(2 diastereomers)

1658 mg of alcohol A (2.0 mmol) was dissolved in 10 ml of drydichloromethane under an argon atmosphere. The solution was treated with1.03 ml of N-ethyldiisopropylamine (6.0 mmol) and 0.63 ml of monoallyln-diisopropylchlborphosphoramidite (2.8 mmol) and stirred for 1 h atroom temperature. The excess phosphorylation reagent was then destroyedby addition of 61 μl (0.8 mmol) of isopropanol. After 10 min, themixture was concentrated in vacuo and the residue was purified by flashchromatography on 3.3×21 cm silica gel using hexane/EtOAc/NEt₃(75:25:1). The product-containing fractions were concentrated, taken upin CCl₄ and concentrated again. 2.04 g of an almost colourless foam(quant.) were obtained, which can be used thus directly f oroligomerization and can be kept at −20° C. for a number of weeks .

-   TLC on silica gel (EtOAc/hexane/NEt₃ 33:66:1): 0.41

¹H-NMR (CDCl₃, 300 MHz): selected characteristic positions: 2.42, 2.53,(2 dd, J=5.0, 11.0 Hz, 2H, 2 H-5′eq), 3.76, 3.77, 3.78, 3.79 (4 s, 4×3H,OMe), 5.70, 5.73 (2 d, J=9.0 Hz, 2H, 2H-1′), 6.16, 6.29 (2 bs, 2H, 2H-3′)

³¹P-NMR (CDCl₃): 150.6, 151.0

5.3 Lysine-based Linker

The synthesis is depicted in Scheme 7 and is described in detail below.

6-Amino-2(S)-hydroxyhexanoic acid (1) was prepared from L-lysine in amanner known from the literature by diazotization and subsequenthydrolysis (K.-I. Aketa, Chem. Pharm Bull. 1976, 24, 621).

2-(S)-N-Fmoc-6-amino-1,2-hexanediol (2)

3.4 g of LiBH₄ (156 mmol, 4 eq) are dissolved under argon in 100 ml ofabs. THF (exothermic!). After cooling to about 30° C., 39.6 ml of TMSCl(312 mmol, 8 eq) are slowly added dropwise (evolution of gas!), aprecipitate being formed. 5.74 g of 6-amino-2(S)-hydroxyhexanoic acid(1) (39 mmol) are added in portions in an argon countercurrent and themixture is heated to 65° C. until the TLC (silica gel; i-PrOH/conc.NH₄OH/water 7:2:1; staining with ninhydrin) no longer shows any startingmaterial (about 3 h). The mixture is cautiously treated with 120 ml ofmethanol with ice-cooling (strong evolution of gas!). The solvent isconcentrated in vacuo, and the residue is co-evaporated three times with200 ml of methanol each time and then dissolved in 100 ml of abs. DMF.After addition of 16 ml of ethyldiisopropylamine (93.6 mmol, 2.4 eq),the mixture is cooled to 0° C. and treated; in portions with 12.1 g ofFmocCl (46.8 mmol, 1.2 eq) After 15 minutes, the cooling bath is removedand the mixture is stirred at room temperature until the starting:material has been consumed (about 3 h; TLC checking: silica gel;CHCl₃/MeOH/HOAc/water 060:30:3:5) The reaction solution is added to 600ml of satd. NaHCO₃ solution. The precipitate is filtered off, washedwith 200 ml of water and dried at 50° C. in high vacuum until the weightis constant (about 6 h). 13.9 g of a colourless solid is obtained, whichis recrystallized from ethyl acetate (40 ml)/n-hexane (35 ml). Yield:9.05 g (65%).

¹H-NMR (300 MHz, CDCl₃): 7.68, 7.51 (2 d, J=8.0 Hz, in each case 2H,Ar—H), 7.32 (t, J=8.0 Hz, 2H, Ar—H), 7.23 (dt, J=1.6, 8.0 Hz, 2H, Ar—H),4.92 (bs, 1H, NH), 4.32 (d, J=7 Hz, 2H, OCOCH₂), 4.13 (bt, J=7.0 Hz,OCOCH₂CH), 3.64–3.58 (m, 1H, H-1, H-1′, H-2, H-6, H-6′), 3.54 (dd,J=3.2, 11.0 Hz, 1H, H-1, H-1′, H-2, H-6, H-6′), 3.35 (dd, J=7.4, 11.0Hz, 1H, H-1, H-1′, H-2, H-6, H-6′), 3.16–3.06 (m, 2 H H-1, H-1′, H-2,H-6, H-6′), 3.0–2.0 (bs, 2H, OH), 1.52–1.18 (m, 6H, H-3, H-3′, H-4,H-4′, H-5, H-5′).

2-(S)-N-Fmoc-O¹-DMT-6-amino-1,2-hexanediol (3) was DM-tritylatedaccording to WO 89/024392-(S)-N-Fmoc-O¹-DMT-O²-allyloxydiisopropylaminophosphinyl-6-amino-1,2-hexanediol(4)

0.51 ml of ethyldiisopropylamine (3.0 mmol, 3 eq) and 0.33 ml ofchloro-N,N-diisopropylaminoallyloxyphosphine (1.5 mmol, 1.5 eq) areadded under argon to a solution of 670 mg of the alcohol (3) (1.02 mmol)in 10 ml of abs. dichloromethane. The mixture is stirred at roomtemperature for 2 h, the solvent is stripped off in vacuo and theresidue obtained 1 is purified by flash chromatography on 3.2×16 cmsilica gel (EtOAc/isohexane/NEt₃ 20:80:1). 839 mg (97%) of a slightlyyellowish oil are obtained.

-   TLC: silica gel; EtOAc/isohexane/NEt₃ 50:50:1; UV; R_(f)=0.77.

¹H=NMR (300 MHz, CDCl₃): 7.70–6.68 (m, 21H, Ar—H), 4.92–4.62 (m, 1H,NH), 4.31 (d, J=7.0 Hz, H, OCOCH₂), 4.13 (t, J=7.0 Hz, 1H, OCOCH₂CH),3.98–3.40 (m, 5H), 3.77 (2 s, in each case 3H, OMe), 3.16–2.86 (m, 4H),2.58 (t, J=7.0 Hz, 1H, CHCN), 2.38 (t, 1H, CHCN), 1.80–1.20 (m, 6 H),1.20, 1.18, 1.17, 1.16, 1.15, 1.13, 1.08, 1.06 (8 s, 12H, NMe).

³¹P-NMR (300 MHz, CDCl₃): 149.5, 149.0 (2 s)

Example 6 Synthesis of a p-RNA Oligo of the Sequence 4′-IndoleLinker-A8-2′ Using Benzimidazolium Triflate as a Coupling Reagent

108 mg of indole linker phosphoramidite and 244 mg of sphoramidite; areweighed into a synthesizer vial and left in a high vacuum for 3 h in adesiccator over KOH together with the column packed with 28.1 mg of CDPsupport, loaded with A unit. The phosphoramidites are dissolved 1 ml(indole linker) or 2.5 ml (A phosphoramidite) of acetonitrile and a fewbeads of the molecular sieve are added and left closed in a desiccatorover KOH.

200 mg of iodine are dissolved in 50 ml of acetonitrile with vigorousstirring. After everything has dissolved (visual control), 23 ml ofwater and 4.6 ml of symcollidine are added and the solution isthoroughly mixed once. For detritylation, a 6% strength solution ofdichloroacetic acid in dichloromethane is employed. The capping reagent(acetic anhydride+base) is purchased and used as customary foroligonucleotide synthesis.Benzimidazolium triflate is recrystallized from hot acetonitrle anddried. Using the almost colourless crystals, a 0.1 M solution inanhydrous acetonitrile is prepared as a cuping reagent. During thesynthesis, this solution always remains clear and no blockages in thesynthesizer tubing occur.Modified DNA coupling cycling the Eppendorf Ecosyn 300+ (DMT-one):

Detritylation 7 minutes Coupling 1 hour Capping 1.5 minutes Oxidation 1minute20 mg of tetrakis(triphenylphosphine)palladium is dissolved in 1.5 ml ofdichloromethane, 20 mg of diethylammonium hydrogencarbonate, 20 mg oftriphenylphosphine and the glass support carrying the oligonucleotideare added, tightly sealed (parafilm) and the vial is agitated for 5 h atRT. The glass support is then filtered off with suction by means of ananalytical suction filter, and washed with dichloromethane, with acetoneand with water.

The support is suspended using aqueous 0.1 molar sodiumdiethyldithiocarbamate solution and left at RT. For 45 min. It isfiltered off with suction, and washed with water, acetone, ethanol anddichloromethane. The support is suspended in 1.5 ml of 24% strengthhydrazine hydrate solution, shaken for, 24–36 h at 4° C. and diluted to7 ml with 0.1 molar triethylammonium hydrogencarbonate buffer (TEABbuffer). It was washed until hydrazine-free by means of a Waters Sep-Pakcartridge. It is treated with 5 ml of an 80% strength formic acidsolution, and concentrated to dryness after 30 min. The residue is takenup in 10 ml of water, extracted with dichloromethane, and the aqueousphase is concentrated and then HPL chromatographed (tR=33 min, gradientof acetonitrile in 0.1M triethylammonium acetate buffer). Customarydesalting (Waters Sep-Pak cartridge) yields the oligonucleotide.

Yield: 17.6 OD

Substance identity proved by ESI mass spectroscopy:

M(calc.)=3082 D, (M+2H)²⁺(found)=1541.9 D.

Example 7 Preparation of Conjugates 1. Sequential Process

A p-RNA oligomer of the sequence A₈, i.e. an octamer, is first preparedon the Eppendorf Ecosyn D 300+ as described in Example 2 and thefollowing reagents are then exchanged: 6% strength dichloroacetic acidfor 2% strength trichloroacetic acid, iodine in collidine for iodine inpyridine, benzimidazolium triflate solution for tetrazole solution.After changing the synthesis programme, a DNA oligomer of the sequenceGATTC is further synthesized according to known methods (M. J. Gait,Oligonucleotide Synthesis, IRL Press, Oxford, UK 1984). Thedeallylation, hydrazinolysis, HPL chromatography and desalting iscarried out as described for the p-RNA oligomer (see above) and yieldsthe desired conjugate.

2. Convergent Process

As described in Example 2, a p-RNA oligomer having the sequence4′-indole linker-A₈-2′ is prepared, purified, and iodoacetylated. A DNAoligomer of the sequence GATTC-thiol linker is synthesized according toknown methods (M. J. Gait, Oligonucleotide Synthesis, IRL Press, Oxford,UK 1984) and purified (3′-thiol linker from Glen Research: No. 20-2933).On allowing the two fragments to stand (T. Zhu et al., Bioconjug. Chem.1994, 5, 312) in buffered solution, the conjugate results, which isfinally purified by means of HPLC.

Example 8 Conjugation of a Biotin Radical to an Amino-modified p-RNA

First, analogously to the procedure described in Example 6, a p-RNAoligomer of the sequence TAGGCAAT, which is provided with an amino groupat the 4′-end by means of the 5′-amino modifier 5 of Eurogentec(2-(2-(4-monomethoxytrityl)aminoethoxy)ethyl 2-cyanoethyl(N,N-diisopropyl)phosphoramidite), was synthesized and worked up. Theoligonucleotide (17.4 OD, 0.175 μmol) was taken up in 0.5 ml of basicbuffer, 1.14 mg (2.5 μmol) of biotin-N-hydroxysuccinimide ester weredissolved in 114 μl of DMF (abs.) and the solution was allowed to standat RT for 1 h. The resulting conjugate was purified by means ofpreparative HPLC and the pure product was desalted using a Sepak.

Yield: 8.6 OD (49%)

M (calc.)=3080 D, M (eff.)=3080.4 D

Example 9 Preparation of Cyanine or Biotin-labelled p-RNA Online

The various A,T,G,C and Ind (Ind=aminoethylindole as a nucleobase)phosphoramidites were first prepared according to known processes.Cyanine (Cy3-CE) and biotin phosphoramidite were obtained from GlenResearch.

The fully automatic solid-phase synthesis was carried out using 15 μmolin each case. One synthesis cycle consists of the following steps

-   (a) Detritylation: 5 minutes with 6% DCA (dichloroacetic acid) in    CH₂Cl₂ (79 ml);-   (b) Washing with CH₂Cl₂ (20 ml), acetonitrile (20 ml) and then    flushing with argon;-   (c) Coupling: washing of the resin with the activator (0.5 M    pyridine.HCl in CH₂Cl₂, 0.2 ml; 30 minutes' treatment with activator    (0.76 ml) and corresponding phosphoramidite (0.76 ml: 8 eq; 0.1 M in    acetonitrile) in the ratio 1:1;-   (d) Capping: 2 minutes with 50% Cap A (10.5 ml) and 50% Cap B    (10.5 ml) from Perseptive (Cap A: THF, lutidine, acetic anhydride;    Cap B: 1-methylimidazole, THF, pyridine).-   (e) Oxidation: 1 minute with 120 ml of iodine solution (400 mg of    iodine in 100 ml of acetonitrile, 46 ml of H₂O and 9.2 ml of    sym-collidine).-   (f) Washing with acetonitrile (22 ml).

To facilitate the subsequent HPLC purification of the oligonucleotides,the last DMT (dimethoxytrityl) or MMT (monomethoxytrityl) protectivegroup was not removed from biotin or cyanine monomers. The detection ofthe last coupling with the modified phorphoramidites was carried outafter the synthesis with 1% of the resin by means of a trityl cationabsorption in UV (503 nm).

Work-up of the Oligonucleotide

The allyl ether protective groups were removed with a solution oftetrakis(triphenylphosphine)palladium (272 mg), triphenylphosphine (272mg) and diethylammonium hydrogencarbonate in CH₂Cl₂ (15 ml) after 5hours at RT. The glass supports were then washed with CH₂Cl₂ (30 ml),acetone (30 ml) and water (30 ml). In order to remove palladium complexresidues, the resin was rinsed with an aqueous 0.1 M sodiumdiethyldithiocarbamate hydrate solution. The abovementioned washingoperation was carried out once more in a reverse order. The resin wasthen dried in a high vacuum for 10 minutes. The removal step from theglass support with simultaneous debenzoylation was carried out in 24%hydrazine hydrate solution (6 ml) at 4° C. After HPLC checking on RP 18(18–25 hours), the oligonucleotide “Trityl ON” was freed from thehydrazine by means of an activated (acetonitrile, 20 ml) Waters Sep-PakCartridge. The hydrazine was washed with TEAB 0.1 M (30 ml). Theoligonucleotide was then eluted with acetonitrile/TEAB, 0.1 M (10 ml).The mixture was then purified by means of HPLC (for the separation offragment sequences) and the DMT deprotection (30 ml of 80% strengthaqueous formic acid) was carried out. Final desalting (by means ofSep-Pak Cartridge, with TEAB buffer 0.1 M/acetonitrile: 1/1) yielded thepure cyanine- or biotin-labelled oligomers.

An aliquot of this oligo solution was used for carrying out an ESI-MS.

4′ Cy-AIndTTCCTA 2′: calculated M=3026,

found (M+H)⁺=3027.

4′ Biotin-TAGGAAIndT 2′: calculated M=3014,

found (M+2H)²⁺ m/e 1508 and (M+H)⁺ m/e 3015.

The oligos were freeze-dried for storage.

Example 10 Iodoacetylation of p-RNA with N-(iodoacetyloxy)-succinimide

p-RNA sequence: 4′ AGGCAIndT 2′ M_(w)=2266.56 g/mol(Ind=indole-CH₂—CH₂—NH₂-linker)

1 eq. of the p-RNA was dissolved (1 ml per 350 nmol) in a 0.1 molarsodium hydrogencarbonate solution (pH 8.4) and treated (40 μl per mg)with a solution of N-(iodoacetyloxy)succinimide in DMSO. The batch wasblacked out with aluminium film and it was allowed to stand at roomtemperature for 30–90 minutes.

The progress of the reaction was monitored by means of analytical HPLC.The standard conditions are:

Buffer A: 0.1 molar triethylammonium acetate buffer in water

Buffer B: 0.1 molar triethylammonium acetate buffer inwater:acetonitrile 1:4

Gradient: starting from 10% B to 50% B in 40 minutes

Column material: 10 μM LiChrosphere® 100 RP-18 from Merck DarmstadtGmbH; 250×4 mm

Retention time of the starting materials: 18.4 minutes

Retention time of the products in this case: 23.1 minutes

After reaction was complete, the batch was diluted to four times thevolume with water. A Waters Sep-Pak Cartridge RP-18 (from 15 OD 2 g ofpacking) was activated with 2×10 ml of acetonitrile and 2×10 ml ofwater, the oligo was applied and allowed to sink in, and the reactionvessel was washed with 2×10 ml of water, rewashed with 3×10 ml of waterin order to remove salt and reagent, and eluted first with 5×1 ml of50:1 water: acetonitrile and then with 1:1 water:acetonitrile. Theproduct eluted in the 1:1 fractions in very good purity. The fractionswere concentrated in the cold and in the dark, combined, andconcentrated again.

The yields were determined by means of UV absorption spectrometry at 260nm.

Mass spectrometry:

Sequence: 4′ AGGCAInd(CH₂CH₂NHCOCH₂-I)T 2′

calculated mass: 2434.50 g/mol

found mass MH₂ ²⁺: 1217.9 g/mol=2433 g/mol.

Example 11 Conjugation of p-RNA to a Peptide of the Sequence CYSKVG

The iodoacetylated p-RNA (M_(w)=2434.50 g/mol) was dissolved in a buffersystem (1000 μl per 114 nmol) and then treated with a solution of thepeptide in buffer (2 mol of CYSKVG peptide; M_(w)=655.773 g/mol; 228nmol in 20 μl of buffer).

Buffer system: Borax/HCl buffer from Riedel-de Haën, pH 8.0, was mixedin the ratio 1:1 with a 10 millimolar solution of EDTA disodium salt inwater and adjusted to pH 6.3 using HCl. A solution was obtained by thismeans which contained 5 mM Na₂EDTA.

The batch was left at room temperature in the dark until conversion wascomplete. The reaction was monitored by means of HPLC analysis.

The standard conditions are:

Buffer A: 0.1 molar triethylammonium acetate buffer in water

Buffer B: 0.1 molar triethylammonium acetate buffer inwater:acetonitrile 1:4

Gradient: starting from 10% B to 50% B in 40 minutes

Column material: 10 μM LiChrosphere® 100 RP-18 from Merck DarmstadtGmbH; 250×4

Retention time of the starting material: 17.6 minutes

Retention time of the product: 15.5 minutes

After reaction was complete the batch was purified directly by means ofRP-HPLC.

(Standard conditions see above).

The fractions were concentrated in the cold and in the dark, combinedand concentrated again. The residue was taken up in water and desalted.A Waters Sep-Pak Cartridge RP-18 (from 15 D 2 g of packing) wasactivated with 2×10 ml of acetonitrile and 2×10 ml of water, the oligowas applied and allowed to sink in, and the reaction vessel was washedwith 2×10 ml of water, rewashed with 3×10 ml of water in order to removethe salt, and eluted with water: acetonitrile 1:1. The product fractionswere concentrated, combined, and concentrated again.

The yields were determined by means of UV absorption spectrometry at 260nm. They reached 70–95% of theory.

Mass spectrometry:

Sequence: 4′ AGGCAInd(CH₂CH₂NHCOCH₂-CYSKVG)T 2′

calculated mass: 2962.36 g/mol

found mass MH₂ ²⁺: 1482.0 g/mol=2962 g/mol.

Example 12 Conjugation of p-RNA to a Peptide Library

The iodoacetylated p-RNA (M_(w)=2434.50 g/mol was dissolved (1300 μl per832 nmol) in a buffer system and then treated in buffer (8 mol; meanmolecular mass M_(m)=677.82 g/mol; 4.5 mg=6.66 μmol in 200 μl of buffer)with a solution of the peptide library (CKR-XX-OH); X=Arg, Asn, Glu,His, Leu, Lys, Phe, Ser, Trp, Tyr).

Buffer system: Borax/HCl buffer from Riedel-de Haen, pH 8.0, was mixedin the ratio 1:1 with a 10 millimolar solution of EDTA disodium salt inwater and adjusted to pH 6.6 using HCl. A solution was obtained by thismeans which contains 5 mM Na₂EDTA.

The batch was left at room temperature in the dark until conversion wascomplete. The reaction was monitored by means of HPLC analysis. In thiscase, the starting material had disappeared after 70 hours.

The standard conditions of the analytical HPLC are:

Buffer A: 0.1 molar triethylammonium acetate buffer in water

Buffer B: 0.1 molar triethylammonium acetate buffer inwater:acetonitrile 1:4

Gradient: starting from 10% B to 50% B in 40 minutes

Column material: 10 μM LiChrosphere® 100 RP-18 from Merck DarmstadtGmbH; 250×4

Retention time of the starting material: 18.8 minutes

Retention time of the product: several peaks from 13.9–36.2 minutes

After reaction was complete, the batch was diluted to four times thevolume using water. A Waters Sep-Pak Cartridge RP-18 (from 15 OD 2 g ofpacking) was activated with 3×10 ml of acetonitrile and 3×10 ml ofwater, the oligo was applied and allowed to sink in, the reaction vesselwas rewashed with 2×10 ml of water, and the cartridge was rewashed with3×10 ml of water in order to remove salt and excess peptide, and elutedwith 1:1 water: acetonitrile until no more product eluted by UVspectroscopy. The fractions were concentrated in the cold and in thedark, combined, and concentrated again.

1. A conjugate comprising a 4′,2′-pentopyranosylnucleic acid covalentlybonded through a linker to a biomolecule selected from the groupconsisting of a peptide, protein, nucleic acid, antibody, functionalantibody moiety, DNA, and RNA, wherein the biomolecule does notnon-covalently bond or hybridize to the pentopyranosylnucleic acid. 2.The conjugate according to claim 1, wherein the biomolecule is apeptide, protein or a nucleic acid.
 3. The conjugate according to claim1, wherein the biomolecule is an antibody, a functional moiety of anantibody, a DNA, or an RNA.